Aimen F. Shaaban

HUMAN MESENCHYMAL STEM CELLS ENGRAFT AND DEMONSTRATE SITE-SPECIFIC DIFFERENTIATION AFTER IN UTERO TRANSPLANTATION IN SHEEP

Mesenchymal stem cells are multipotent cells that can be isolated from adult bone marrow and can be induced in vitro and in vivo to differentiate into a variety of mesenchymal tissues, including bone, cartilage, tendon, fat, bone marrow stroma, and muscle. Despite their potential clinical utility for cellular and gene therapy, the fate of mesenchymal stem cells after systemic administration is mostly unknown. To address this, we transplanted a well-characterized human mesenchymal stem cell population into fetal sheep early in gestation, before and after the expected development of immunologic competence. In this xenogeneic system, human mesenchymal stem cells engrafted and persisted in multiple tissues for as long as 13 months after transplantation. Transplanted human cells underwent site-specific differentiation into chondrocytes, adipocytes, myocytes and cardiomyocytes, bone marrow stromal cells and thymic stroma. Unexpectedly, there was long-term engraftment even when cells were transplanted after the expected development of immunocompetence. Thus, mesenchymal stem cells maintain their multipotential capacity after transplantation, and seem to have unique immunologic characteristics that allow persistence in a xenogeneic environment. Our data support the possibility of the transplantability of mesenchymal stem cells and their potential utility in tissue engineering, and cellular and gene therapy applications.

Immunosuppressive CD71+ erythroid cells compromise neonatal host defence against infection

Newborn infants are highly susceptible to infection. This defect in host defence has generally been ascribed to the immaturity of neonatal immune cells; however, the degree of hyporesponsiveness is highly variable and depends on the stimulation conditions. These discordant responses illustrate the need for a more unified explanation for why immunity is compromised in neonates. Here we show that physiologically enriched CD71+ erythroid cells in neonatal mice and human cord blood have distinctive immunosuppressive properties. The production of innate immune protective cytokines by adult cells is diminished after transfer to neonatal mice or after co-culture with neonatal splenocytes. Neonatal CD71+ cells express the enzyme arginase-2, and arginase activity is essential for the immunosuppressive properties of these cells because molecular inhibition of this enzyme or supplementation with l-arginine overrides immunosuppression. In addition, the ablation of CD71+ cells in neonatal mice, or the decline in number of these cells as postnatal development progresses parallels the loss of suppression, and restored resistance to the perinatal pathogens Listeria monocytogenes and Escherichia coli. However, CD71+ cell-mediated susceptibility to infection is counterbalanced by CD71+ cell-mediated protection against aberrant immune cell activation in the intestine, where colonization with commensal microorganisms occurs swiftly after parturition. Conversely, circumventing such colonization by using antimicrobials or gnotobiotic germ-free mice overrides these protective benefits. Thus, CD71+ cells quench the excessive inflammation induced by abrupt colonization with commensal microorganisms after parturition. This finding challenges the idea that the susceptibility of neonates to infection reflects immune-cell-intrinsic defects and instead highlights processes that are developmentally more essential and inadvertently mitigate innate immune protection. We anticipate that these results will spark renewed investigation into the need for immunosuppression in neonates, as well as improved strategies for augmenting host defence in this vulnerable population.

Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI

BackgroundA number of gene therapy applications would benefit from vectors capable of expressing multiple genes. In this study we explored the feasibility and efficiency of expressing two or three transgenes in HIV-1 based lentiviral vector. Bicistronic and tricistronic self-inactivating lentiviral vectors were constructed employing the internal ribosomal entry site (IRES) sequence of encephalomyocarditis virus (EMCV) and/or foot-and-mouth disease virus (FMDV) cleavage factor 2A. We employed enhanced green fluorescent protein (eGFP), O6-methylguanine-DNA-methyltransferase (MGMT), and homeobox transcription factor HOXB4 as model genes and their expression was detected by appropriate methods including fluorescence microscopy, flow cytometry, immunocytochemistry, biochemical assay, and western blotting.ResultsAll the multigene vectors produced high titer virus and were able to simultaneously express two or three transgenes in transduced cells. However, the level of expression of individual transgenes varied depending on: the transgene itself; its position within the construct; the total number of transgenes expressed; the strategy used for multigene expression and the average copy number of pro-viral insertions. Notably, at limiting MOI, the expression of eGFP in a bicistronic vector based on 2A was ~4 times greater than that of an IRES based vector.ConclusionThe small and efficient 2A sequence can be used alone or in combination with an IRES for the construction of multicistronic lentiviral vectors which can express encoded transgenes at functionally relevant levels in cells containing an average of one pro-viral insert.

In utero bone marrow transplantation induces donor-specific tolerance by a combination of clonal deletion and clonal anergy

BACKGROUND/PURPOSE In utero bone marrow transplantation can induce donor-specific tolerance to postnatal solid organ transplantation, although the mechanisms remain poorly defined. In this study, we investigated the role of clonal deletion and clonal anergy in the maintenance of tolerance in a murine model of in utero bone marrow transplantation. METHODS DBA/2 mice (MIs(a+)) were used as donors of adult bone marrow, and 14-day-gestation fetal Balb/c mice (MIs(a-)) were used as recipients. Tolerance was defined by donor-specific skin graft survival for more than 8 weeks. Clonal deletion was assessed by flow cytometry for Vbeta6 T cell receptor usage. A tolerant animal demonstrating partial deletion of CD4+/Vbeta6+ T cells and a nontolerant animal were selected for analysis of clonal anergy by a proliferation assay using plate-bound anti-Vbeta6 antibody for stimulation with or without exogenous interleukin-2 (IL2). RESULTS Vbeta6+ splenocytes constituted 6.32% of CD4+ T cells in the tolerant animal compared with 9.19% in the nontolerant animal, demonstrating incomplete clonal deletion in the tolerant animal. Stimulation with plate-bound anti-Vbeta6 induced a good proliferative response in the nontolerant animal but a significantly attenuated response in the tolerant animal (P< .001), which was abrogated by the addition of IL2. CONCLUSIONS In this murine model of in utero bone marrow transplantation, the tolerant state is characterized by partial clonal deletion of donor reactive T cells and clonal anergy of nondeleted donor reactive T cells. The anergic state can be abrogated by exogenous IL2, suggesting that the mechanism of anergy is a deficiency of IL2 production.

Age-Related Differences in Diagnosis and Morbidity of Intestinal Malrotation

BACKGROUND Intestinal malrotation in adulthood may present with a variety of chronic symptoms. Surgical intervention frequently leads to other complications in these patients. We hypothesized that the chronic nature of malrotation in adults could cause a delay in diagnosis and increased perioperative complications. STUDY DESIGN All patients diagnosed with intestinal malrotation from July 2002 through July 2006 were included. IRB approval was obtained. Outcomes in patients less than 16 years of age were compared with outcomes from those older than 16. Presenting symptoms, initial diagnosis, results of imaging data, and time to diagnosis were evaluated. Surgical management, resulting complications, and rate of reoperation were analyzed. RESULTS Twenty-four patients with intestinal malrotation were identified (age range, 10 days to 89 years old; 10 adults, 14 children). Seventy percent of adults experienced chronic symptoms for 6 months or more before the diagnosis of malrotation was made (children, 14%, p = 0.017). No patients in the adult group were initially diagnosed with malrotation, although 57% of children were correctly diagnosed at the time of presentation of symptoms (p=0.006). Postoperative complications occurred in 60% of adults, but in only 29% of children, though this did not reach significance (p=0.211). Forty percent of adult patients required reoperation (p=0.020). CONCLUSIONS Intestinal malrotation in adults is often associated with a delay in diagnosis and increased morbidity. Enhanced awareness of this entity in adults may enhance patient counseling and improve therapeutic outcomes in these patients.

Cross-Generational Reproductive Fitness Enforced by Microchimeric Maternal Cells

Exposure to maternal tissue during in utero development imprints tolerance to immunologically foreign non-inherited maternal antigens (NIMA) that persists into adulthood. The biological advantage of this tolerance, conserved across mammalian species, remains unclear. Here, we show maternal cells that establish microchimerism in female offspring during development promote systemic accumulation of immune suppressive regulatory T cells (Tregs) with NIMA specificity. NIMA-specific Tregs expand during pregnancies sired by males expressing alloantigens with overlapping NIMA specificity, thereby averting fetal wastage triggered by prenatal infection and non-infectious disruptions of fetal tolerance. Therefore, exposure to NIMA selectively enhances reproductive success in second-generation females carrying embryos with overlapping paternally inherited antigens. These findings demonstrate that genetic fitness, canonically thought to be restricted to Mendelian inheritance, is enhanced in female placental mammals through vertically transferred maternal cells that promote conservation of NIMA and enforce cross-generational reproductive benefits.

Selection, enrichment, and culture expansion of murine mesenchymal progenitor cells by retroviral transduction of cycling adherent bone marrow cells.

It has been difficult to characterize murine bone marrow (BM)-derived mesenchymal progenitor cells (MPCs) because of contamination with hematopoietic cells. We took advantage of the rapid proliferation of MPCs after replating to enrich murine MPCs by transfection with a retroviral vector carrying both LacZ and the selective neomycin resistance (neoR) gene. Freshly harvested BM cells from mice were incubated with BAG retroviral vector produced by amphotropic psi-CRIP or ecotropic psi-CRE producer cells for 48 hours and grown in the presence of G418.Cells incubated in psi-CRIP supernatant formed colonies composed of large homogeneous cells that were free of CD45(+) cells, but cells incubated in psi-CRE supernatant did not form stromal cell colonies. In the undifferentiated state, the cells displayed a fibroblast-like phenotype with low alkaline phosphatase activity. However, upon treatment with dexamethasone or 5-azacytidine, the retrovirally transduced cells differentiated into oil-red-O-positive adipocytic cells and osteogenic cells generating von Kossa-positive bone nodules. Osteogenic supplements composed of beta-glycerophosphate, dexamethasone, and ascorbic acid induced an increase in alkaline phosphatase activity and acute osteogenesis associated with early cell detachment. Subcutaneous injection with retrovirally transduced cells into day 1 newborn mice of the same strain produced ectopic calcium depositions surrounded by X-gal(+) cells. Retroviral selection of cycling adherent cells is an effective approach for enrichment of MPCs.

Early laparoscopic fundoplication and gastrostomy in infants with spinal muscular atrophy type I

BACKGROUND/PURPOSE Spinal muscular atrophy (SMA) in children leads to progressive muscle weakness, dysphagia, aspiration, and death. We hypothesized that early laparoscopic fundoplication and gastrostomy in infants with SMA type I could be performed safely perhaps leading to fewer aspiration events and improved nutritional status. METHODS Children diagnosed with SMA type I from 2002 through 2005 were included (n = 12). All children underwent laparoscopic Nissen fundoplication with gastrostomy shortly after diagnosis. Postoperative respiratory management and discharge criteria were standardized. RESULTS All patients were extubated immediately postoperatively. There were no significant complications. Average time to full feeding and inpatient length of stay were 42 +/- 4.9 hours (range, 30-48 hours) and 78 +/- 22.5 hours (range, 44-120 hours), respectively. Mean weight-for-length percentile was doubled at 1 year postoperatively (P = .03). The number of respiratory-related hospitalizations in the cohort decreased by almost 50% in the ensuing 12 months after surgery, although this did not reach statistical significance in this small cohort (P = .34). CONCLUSIONS Early laparoscopic fundoplication and gastrostomy is safe and is associated with improved nutritional status. A trend toward fewer significant long-term aspiration-related events was seen after fundoplication. To better assess the long-term benefits of performing an antireflux procedure in these high-risk patients, a larger prospective trial comparing current nutritional support practices is needed.

Ultrasound of a torsed ovary: Characteristic gray-scale appearance despite normal arterial and venous flow on Doppler

AbstractBackground. We present the case of an 8-year-old girl with acute onset of intermittent lower abdominal pain. The gray-scale US examination showed an enlarged right ovary with peripheral cysts, reflecting ovarian congestion and strongly suggesting the diagnosis of torsion. Normal arterial and venous flow, however, was found on Doppler US. Objective. To demonstrate the importance of gray-scale US findings despite the presence of blood flow found on Doppler US in salvaging a viable, torsed ovary. Methods. Despite the Doppler findings, a presumptive diagnosis of ovarian torsion was made. Results. Surgery confirmed the presence of a torsed ovary, which was viable and appeared normal after detorsion. Conclusion. This case illustrates that the gray-scale US appearance of the ovary can be more reliable than Doppler US for the diagnosis of ovarian torsion.

Outcomes of plastic closure in gastroschisis.

BACKGROUND Gastroschisis is a congenital abdominal wall defect in which the intestines develop outside the abdomen and are exposed to amniotic fluid. When the defect is small, lymphatic, venous, and intestinal obstruction may occur and contribute to the formation of intestinal edema, atresia, ischemia, and a thick inflammatory peel. Treatment requires early coverage of abdominal contents either by primary closure or by the placement of temporary Silastic silo followed by abdominal wall closure. Currently, both traditional suture closure and the sutureless plastic closure are being employed to repair the gastroschisis defect. The goal of the current study is to evaluate plastic closure. We predict no difference will be found in clinical outcomes between plastic closure and traditional suture closure. METHODS A retrospective review of 80 patients treated between 2000 and 2009 was performed. Plastic closure was used in 52 (65%) and traditional suture closure in 28 (35%) babies. The surgical procedure was determined by surgeon preference. Of the 31(39%) babies who required silos, 15 (19%) were treated with plastic closure and 16 (20%) underwent traditional closure. We collected the following demographic data and clinical progression data. Using SAS 9.2 (SAS Institute Inc, Cary, NC), we conducted linear regression, logistic regression, and time to event models to compare the following outcomes: days on ventilator, days to start enteral feeds, days to reach goal enteral feeds, days on total parenteral nutrition, hospital charges, duration of stay, mortality, and complications. RESULTS The mean duration of follow-up was 11.4 months. Patients spent an average of 6 days on the ventilator. There were 2 mortalities. A multivariate analysis demonstrated that no differences were found between the 2 closures with most of the outcomes; however, when compared with traditional suture closure, those babies treated with plastic closure spent 4 days fewer days on the ventilator (P < .01). Those babies who underwent suture closure were more likely to have an infection or sepsis (odds ratio, 5.15; P < .001). When the entire cohort was considered, no significant difference was found between plastic and suture closure in time to start feeds, time to reach goal feeds, time on parenteral nutrition, hospital charges, duration of stay, or complications. Ventral hernias were noted in 46 (58%) patients, 32 (62%) after plastic closure and 14 (50%) after suture closure (P = .32). Hernia repair was required in 16 (20%) patients, 11 (21%) after plastic closure, and 5 (18%) after traditional repair (P = .32). In the silo cohort, children treated with plastic closure required 7.5(P < .01) fewer days to start enteral feeds than those treated with suture closure. CONCLUSION Plastic closure of abdominal wall defects in gastroschisis is effective both as a primary procedure and after silo placement. A multivariate analysis shows plastic closure to be associated with fewer days of mechanical ventilation and less likelihood of developing infection or sepsis.