Congenital defects among liveborn infants with Down syndrome

BACKGROUND: Many infants with Down syndrome (DS) have co-occurring congenital malformations requiring intensive surgical and medical management. To anticipate the care needed by these infants, providers and parents require accurate information about birth defects that may be present. This article uses a unique national hospital discharge dataset to identify the rate at which structural birth defects are identified among liveborn infants with DS. METHODS: ICD-9-CM diagnosis codes for data from the Healthcare Cost and Utilization Project were used to identify infants with and without DS, and to classify birth defects. The study population consisted of liveborn infants discharged from the hospital from 1993 through 2002. ORs for the association between the occurrence of congenital malformations and the presence of DS were computed using logistic regression models for survey data. RESULTS: Discharge data included 11,372 DS and 7,884,209 non-DS births, representing national estimates of 43,463 DS and 39,716,469 non-DS births respectively. In addition to congenital heart defects that co-occurred most often in DS infants compared to infants without DS, the risks for gastrointestinal malformations (OR 67.07), genitourinary malformations (OR 3.62), orofacial malformations (OR 5.63), and abdominal wall malformations (OR 3.25) were also elevated in infants with DS. There was no difference in the risk of spina bifida between infants with and without DS. CONCLUSIONS: This is the first nationally representative compilation of the co-occurrence of congenital malformations associated with DS. This information may assist providers and parents in their attempts to understand and prepare for the true burden of this condition.     

Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing

Several recent studies suggest the isolation of stem cells in skeletal muscle, but the functional properties of these muscle-derived stem cells is still unclear. In the present study, we report the purification of muscle-derived stem cells from the mdx mouse, an animal model for Duchenne muscular dystrophy. We show that enrichment of desmin(+) cells using the preplate technique from mouse primary muscle cell culture also enriches a cell population expressing CD34 and Bcl-2. The CD34(+) cells and Bcl-2(+) cells were found to reside within the basal lamina, where satellite cells are normally found. Clonal isolation and characterization from this CD34(+)Bcl-2(+) enriched population yielded a putative muscle-derived stem cell, mc13, that is capable of differentiating into both myogenic and osteogenic lineage in vitro and in vivo. The mc13 cells are c-kit and CD45 negative and express: desmin, c-met and MNF, three markers expressed in early myogenic progenitors; Flk-1, a mouse homologue of KDR recently identified in humans as a key marker in hematopoietic cells with stem cell-like characteristics; and Sca-1, a marker for both skeletal muscle and hematopoietic stem cells. Intramuscular, and more importantly, intravenous injection of mc13 cells result in muscle regeneration and partial restoration of dystrophin in mdx mice. Transplantation of mc13 cells engineered to secrete osteogenic protein differentiate in osteogenic lineage and accelerate healing of a skull defect in SCID mice. Taken together, these results suggest the isolation of a population of muscle-derived stem cells capable of improving both muscle regeneration and bone healing.     

Effective treatment of established murine collagen-induced arthritis by systemic administration of dendritic cells genetically modified to express IL-4

Dendritic cells (DC) are APCs that are able to stimulate or inhibit immune responses, depending on levels of expression of MHC class I and II costimulatory molecules and cytokines. Our previous studies have suggested that the observed contralateral effect, where injection of a vector carrying certain immunomodulatory genes into one joint resulted in inhibition of arthritis in untreated joints, is mediated by in vivo modification of DC. Therefore, we have examined the ability of genetically modified DC to suppress established murine collagen-induced arthritis (CIA) after i.v. delivery. IL-4 has been shown to partially reduce the severity of CIA after repeated injection of recombinant protein or by injection of an adenoviral vector expressing IL-4. Here we demonstrate that i.v. injection of immature DC, infected with an adenoviral vector expressing IL-4, into mice with established CIA resulted in almost complete suppression of disease, with no recurrence for up to 4 wk posttreatment. Injection i.v. of fluorescently labeled DC demonstrated that the cells rapidly migrated to the liver and spleen after 6 h and to the lymph nodes by 24 h. In culture, spleen cells from DC/IL-4-treated mice produced less IFN-gamma after stimulation by collagen than did control groups. In addition, DC/IL-4 administration decreased the level of specific Abs against type II collagen, in particular the IgG2 Th1 isotype 14 days posttreatment. These results demonstrate the ability to treat effectively established murine arthritis by systemic administration of DC expressing IL-4.     

Optimization of environmental factors for the production and handling of recombinant retrovirus

Certain steps from the production to infection of the amphotrophic retroviral vector, MFG-LacZ, were optimized and the factors that affect retroviral titers were analyzed. Retroviral vector titers were highest when the culture supernatant was harvested 3 days after the producer cells had reached confluence. About a 2-fold increase in vector production was achieved at 32°C compared to that at 37°C. Low serum concentrations had no significant effect on the titers of virus produced by the CRIP cell line. Retroviral vectors were stable at 4°C but very unstable at 37°C and were quite sensitive to freezing and thawing. About 30%–50% of viral infectivity was lost during the thawing step and the loss was not recovered by the addition of commonly used cryoprotectants. Increase in viral exposure time for infection to target NIH3T3 cells was linearly proportional to the retroviral titer for up to 15 h. In addition, using DEAE-dextran in place of polybrene as a polycation during infection enhanced infection efficiency about 3-fold. The retrovirus was robust to simple ultrafiltration and its titer could be easily concentrated 16-fold. Taken together, our data suggest that at least a 100-fold increase in titer can be achieved with simple optimization.     

Repetitive N-WASP-binding elements of the enterohemorrhagic Escherichia coli effector EspF(U) synergistically activate actin assembly

Enterohemorrhagic Escherichia coli (EHEC) generate F-actin-rich adhesion pedestals by delivering effector proteins into mammalian cells. These effectors include the translocated receptor Tir, along with EspF(U), a protein that associates indirectly with Tir and contains multiple peptide repeats that stimulate actin polymerization. In vitro, the EspF(U) repeat region is capable of binding and activating recombinant derivatives of N-WASP, a host actin nucleation-promoting factor. In spite of the identification of these important bacterial and host factors, the underlying mechanisms of how EHEC so potently exploits the native actin assembly machinery have not been clearly defined. Here we show that Tir and EspF(U) are sufficient for actin pedestal formation in cultured cells. Experimental clustering of Tir-EspF(U) fusion proteins indicates that the central role of the cytoplasmic portion of Tir is to promote clustering of the repeat region of EspF(U). Whereas clustering of a single EspF(U) repeat is sufficient to bind N-WASP and generate pedestals on cultured cells, multi-repeat EspF(U) derivatives promote actin assembly more efficiently. Moreover, the EspF(U) repeats activate a protein complex containing N-WASP and the actin-binding protein WIP in a synergistic fashion in vitro, further suggesting that the repeats cooperate to stimulate actin polymerization in vivo. One explanation for repeat synergy is that simultaneous engagement of multiple N-WASP molecules can enhance its ability to interact with the actin nucleating Arp2/3 complex. These findings define the minimal set of bacterial effectors required for pedestal formation and the elements within those effectors that contribute to actin assembly via N-WASP-Arp2/3-mediated signaling pathways.     

Genetic variation affecting heart rate in Drosophila melanogaster

Heart rate in pre-pupae of Drosophila melanogaster is shown to vary over a wide range from 2.5 to 3.7 beats per second. Quantitative genetic analysis of a sample of 11 highly inbred lines indicates that approaching one-quarter of the total variance in natural populations can be attributed to genetic differences between flies. A hypomorphic allele of the potassium channel gene ether-a-gogo, which is homologous to a human long-QT syndrome susceptibility gene (HERG), has a heart rate at the low end of the wild-type range, but this effect can be suppressed in certain wild-type genetic backgrounds. This study provides a baseline for investigation of pharmacological and other physiological influences on heart rate in the model organism, and implies that quantitative genetic dissection will provide insight into the molecular basis for variation in normal and arrhythmic heart function.     

Viability of Female Germ-Line Cells Homozygous for Zygotic Lethals in DROSOPHILA MELANOGASTER

We have analyzed the viability of different types of X chromosomes in homozygous clones of female germ cells. The chromosomes carried viable mutations, single-cistron zygotic-lethal and semi-lethal mutations, or small (about six chromosome band) deletions. Homozygous germ-line clones were produced by recombination in females heterozygous for an X-linked, dominant, agametic female sterile.

All the zygotic-viable mutants are also viable in germ cells. Of 16 deletions tested (uncovering a total of 93 bands) only 2 (of 4 and 5 bands) are germ-cell viable. Mutations in 15 lethal complementation groups in the zeste-white region were tested. When known, the most extreme alleles at each locus were tested. Only in five loci (33%) were the mutants viable in the germ line. Similar studies of the same deletions and point-mutant lethals in epidermal cells show that 42% of the bands and 77% of the lethal alleles are viable. Thus, germ-line cells have more stringent cell-autonomous genetic requirements than do epidermal cells.

The eggs recovered from clones of three of the germ-cell viable zw mutations gave embryos arrested early in embryogenesis, although genotypically identical embryos derived from heterozygous oogonia die as larvae or even hatch as adult escapers. For two genes, homozygosis of the mutations tested also caused embryonic arrest of heterozygous female embryos, and in one case, the eggs did not develop at all. Germ-line clones of one quite leaky mutation gave eggs that were indistinguishable from normal. The abundance of genes whose products are required for oogenesis, whose products are required in the oocyte, and whose activity is required during zygotic development is discussed.