In utero surgery rescues neurological function at birth in sheep with spina bifida.

We hypothesize that the neurologic deficit associated with open spina bifida is not directly caused by the primary defect but rather is due to chronic mechanical and chemical trauma since the unprotected neural tissue is exposed to the intrauterine environment. We report here that exposure of the normal spinal cord to the amniotic cavity in midgestational sheep fetuses leads to a human-like open spina bifida with paraplegia at birth, indicating that the exposed neural tissue is progressively destroyed during pregnancy. When open spina bifida was repaired in utero at an intermediate stage, the animals had near-normal neurologic function. The spinal cord was deformed but largely preserved. These findings suggest that secondary neural tissue destruction during pregnancy is primarily responsible for the functional loss and that timely in utero repair of open spina bifida might rescue neurologic function.

Allotype suppression in the rabbit. I. The ontogeny of cells bearing immunoglobulin of paternal allotype and the fate of these cells after treatment with antiallotype antisera.

The ability to distinguish immunoglobulin (Ig) of paternal allotype both on lymphocyte membranes and in the serum of neonatal b(4)b(5) heterozygous rabbits has allowed us to study the postnatal ontogeny of cells bearing endogenously synthesized Ig that could not have been derived from the mother. In normal b(4)b(5) rabbits, such endogenously synthesized Ig of paternal allotype is present on the membranes of bone marrow-derived (B) lymphocytes from birth, but does not appear as detectable circulating Ig until approximately 2 wk of age. In the neonate, cells bearing the paternal allotype are potential targets for the induction of chronic allotype suppression. Within 24 h of exposure to anti-allotype antisera in vivo, Ig of the suppressed paternal allotype is no longer detectable on the surface of neonatal lymphoid cells. Further, this lymphocyte membrane Ig is eliminated and not simply masked by the suppressing antibodies. Finally, cells bearing the suppressed allotype are deleted from all lymphoid organs for the duration of total allotype suppression, and reappear first in bone marrow and peripheral blood at the time of spontaneous escape from suppression.

Graft-vs.-host reactions in reciprocal hybrid mice. I. Dissociation of T-cell activities in the mixed lymphocyte reaction and two graft-vs.-host assays.

Spleen cells from BALB/c and C57BL/6 mice were tested for their reactivity against reciprocal hybrid tissues ((BALB/c x C57BL/6) F(1) and (C57BL/6 x BALB/c) F(1)) in three assay systems: the mixed lymphocyte reaction (MLR); the Simonsen spleen-weight graft-vs.-host (GVH) assay; and a GVH mortality assay. It was shown that both F(1)'s serve as equally effective stimulators of parental cells in the MLR. In the spleen-weight assay, BALB/c and C57BL/6 cells were equally active in a given host, but greater splenomegaly was observed in (BALB/c x C57BL/6) F(1) hosts regardless of the donor strain. By contrast, BALB/c cells were much less lethal than C57BL/6 cells in (BALB/c x C57BL/6) F(1) hosts than in (C57BL/6 x BALB/c) F(1) hosts, and to a lesser degree C57BL/6 cells were less lethal than BALB/c cells in (C57BL/6 x BALB/c) F(1) hosts. The possibility that modifying substances may differentially alter reactivity of parental lymphocytes and that considerations other than genotype determine the outcome of a GVH reaction are discussed in detail.

Stimulus-response in the mixed lymphocyte reaction.

Mixed lymphocyte reactions occur when mouse spleen cell populations depleted of thymus-derived (T) lymphocytes are cultured with allogeneic target cells inactivated by mitomycin C or X irradiation, and when F(1) hybrid responder cells are cultured with inactivated parental target cells. These responses might be interpreted as indicating that T lymphocytes are not required for responsiveness and that F(1) lymphocytes recognize parental alloantigens. Data reported here indicate that the more likely explanation for these surprising results is that inactivated target cells recognize the "responding" cells and this recognition leads to the response observed.

Deletion of b5 immunoglobulin-bearing lymphocytes in allotype-suppressed rabbits.

The proportion of peripheral blood lymphocytes with cell surface b5 immunoglobulin (Ig) was compared with serum b5 Ig levels in allotype-suppressed and recovering b5 homozygous rabbits in order to establish the cellular defect in suppression and to determine the relation between lymphocyte membrane Ig and secreted Ig of the same allotype. In rabbits fully suppressed for b5, the absence of circulating b5 Ig is reflected in a complete deletion of b5-bearing lymphocytes. During spontaneous escape from suppression, the appearance of b5-bearing lymphocytes precedes the appearance of detectable serum b5. During recovery from suppression b5-bearing lymphocytes recover rapidly toward normal levels, but circulating b5 levels remain chronically and disproportionately depressed.

Transplantation of fetal hematopoietic stem cells in utero: the creation of hematopoietic chimeras.

Transplantation of normal, immature, fetal hematopoietic cells into a preimmune fetal recipient with a congenital hemoglobinopathy may allow partial reconstitution of normal hemoglobin production without the complications associated with postnatal bone marrow transplantation (immunosuppression and the occurrence of graft versus host disease). In order to test this hypothesis the naturally occurring polymorphism at the beta-hemoglobin locus of the sheep was used as a marker for engraftment and hematopoietic chimerism. Intraperitoneal injection of allogeneic fetal stem cells into normal fetal lambs resulted in hematopoietic chimerism in three of four surviving recipients. This chimerism has been sustained for 6 months after birth and 9 months after engraftment, without evidence of graft versus host disease, and without the use of immunosuppressive therapy.

Fetal spina bifida repair--current trends and prospects of intrauterine neurosurgery.

Myelomeningocele is a common dysraphic defect leading to severe impairment throughout the patient's lifetime. Although surgical closure of this anomaly is usually performed in the early postnatal period, an estimated 330 cases of intrauterine repair have been performed in a few specialized centers worldwide. It was hoped prenatal intervention would improve the prognosis of affected patients, and preliminary findings suggest a reduced incidence of shunt-dependent hydrocephalus, as well as an improvement in hindbrain herniation. However, the expectations for improved neurological outcome have not been fulfilled and not all patients benefit from fetal surgery in the same way. Therefore, a multicenter randomized controlled trial was initiated in the USA to compare intrauterine with conventional postnatal care, in order to establish the procedure-related benefits and risks. The primary study endpoints include the need for shunt at 1 year of age, and fetal and infant mortality. No data from the trial will be published before the final analysis has been completed in 2008, and until then, the number of centers offering intrauterine MMC repair in the USA is limited to 3 in order to prevent the uncontrolled proliferation of new centers offering this procedure. In future, refined, risk-reduced surgical techniques and new treatment options for preterm labor and preterm rupture of the membranes are likely to reduce associated maternal and fetal risks and improve outcome, but further research will be needed.

Engraftment and long-term expression of human fetal hemopoietic stem cells in sheep following transplantation in utero.

Hemopoietic stem cells from human fetal liver were transplanted in utero into preimmune fetal sheep (48-54 days of gestation). The fate of donor cells was followed using karyotype analysis, by immunofluorescence labeling with anti-CD antibodies, and by fluorescent in situ hybridization using human-specific DNA probes. Engraftment occurred in 13 of 33 recipients. Of five live born sheep that exhibited chimerism, all expressed human cells in the marrow, whereas three expressed them in blood as well. Engraftment was multilineage (erythroid, myeloid, and lymphoid) and human hemopoietic progenitors (multipotent colony-forming units, colony-forming units-granulocyte, macrophage, and erythroid burst-forming units) capable of forming colonies in vitro were detected in all five lambs for greater than 2 yr. These progenitors responded to human-specific growth factors both in vitro and in vivo. Thus the administration of recombinant human IL-3 and granulocyte macrophage-colony-stimulating factor to chimeric sheep resulted in a 2.1-3.4-fold increase in the relative expression of donor (human) cells. These results demonstrate that the permissive environment of the preimmune fetal sheep provides suitable conditions for the engraftment and long-term multilineage expression of human hemopoietic stem cells in a large animal model. In this model, donor human cells appear to retain certain phenotypic and functional characteristics that can be used to manipulate the size of donor cell pool.

Subcutaneous endoscopically assisted ligation (SEAL) of the internal ring for repair of inguinal hernias in children: report of a new technique and early results.

BACKGROUND: Open inguinal hernia repair with high ligation is an excellent method of repair in the pediatric population. Advantages of endoscopic repairs include the ability to evaluate the contralateral side, avoidance of access trauma to the vas deferens and gonadal vessels, and decreased operative time. We now report our experience with subcutaneous endoscopically assisted ligation (SEAL),: a novel technique that has proved to be a safe and effective in the treatment of inguinal hernia in the pediatric population. METHODS: The study is based on a retrospective review of 204 pediatric patients with 300 inguinal hernias treated with the SEAL technique from November 2001 to August 2003 at a tertiary referral center. Patient age ranged from 30 days to 16 years at the time of operation, with a mean follow-up of 235 days (median follow-up, 189 days). Statistical analysis was done with chi2 test, with the main outcome measures being intraoperative and postoperative complications including recurrence rate, suture abscesses, and postoperative hydroceles. RESULTS: There were 13 recurrences in 300 SEAL repairs, for a recurrence rate of 4.3% (95% C.I. 2.01%-6.65%), with only two recurrences in the last 100 repairs (2%). There were 10 suture abscesses or granulomas and 7 postoperative hydroceles. There was no statistically significant association between recurrence and gender, age at operation, history of prematurity, bilaterality, or kind of suture used. CONCLUSIONS: Our 4.3% (95% C.I. 2.01-6.65%) recurrence rate is comparable to prior series of laparoscopic repairs citing recurrence rates of 0%-5.7%. The majority of recurrences occurred within the first 4 months of developing this new procedure, with only two recurrences in the last 100 repairs. These pilot data suggest that SEAL is a safe and effective technique for inguinal hernia repair in the pediatric population. A prospective study is planned to compare this laparoscopic technique with open herniorrhaphy.

Hyaluronidase levels in urine from Wilms' tumor patients.

The pathophysiology of Wilms' tumor is associated with major alterations in hyaluronic acid metabolism. Elevated levels of both hyaluronic acid and a hyaluronic acid-stimulating activity occur in the urine and serum of patients with this tumor. In the current study, we describe elevated levels of urinary hyaluronidase in five patients with Wilms' tumor. Following surgical removal of the tumor, enzyme levels decreased toward normal. Characterization of enzyme activity indicates that hyaluronidase may be produced by the tumor itself. Alternatively, normal renal tissue may also be producing enzyme in a compensatory response to the elevated hyaluronic acid levels in these patients. We suggest that urinary hyaluronidase can be used as an additional marker for Wilms' tumor.

Hyaluronic acid-stimulating activity in the pathophysiology of Wilms' tumors.

Markedly elevated levels of hyaluronic acid occur in the serum and urine of some patients with Wilms' tumor. We have recently described a glycoprotein factor in fetal serum that stimulates deposition of hyaluronic acid. In a survey of bovine fetal tissue extracts, we have identified the fetal kidney as the source of this circulating activity. Wilms' tumors arise from transformed "rests" of fetal kidney. We demonstrate here that such tumors continue to produce this fetal factor and that the hyaluronic acid-stimulating activity is found in the urine of children with Wilms' tumors. In the three patients with Wilms' tumor who were followed, elevated levels of hyaluronic acid-stimulating activity were found in their urine before treatment. By 2 months after surgical removal of their tumors, these levels had returned to baseline. We propose that hyaluronic acid-stimulating activity is the mechanism for the elevated levels of hyaluronic acid in the sera and urine of these patients. The activity is an oncofetal protein and the first for which a function has been identified. It also is a marker for this common childhood solid tumor and has the potential for identifying children at increased risk.

Uses and limitations of exercise Doppler echocardiography in the diagnosis of ischemic heart disease.

This study tested the hypothesis that coronary artery disease might be identified by a decrease in Doppler measurements of flow velocity and acceleration. The response of aortic blood flow velocity and acceleration to exercise was determined in 102 subjects (28 young control subjects and 74 older patients) who underwent continuous wave Doppler echocardiographic examination before, during and immediately after near maximal treadmill exercise. Patients were grouped according to the results of thallium perfusion imaging: Group I = normal, Group II = ischemia with or without prior infarction and Group III = prior infarction only. A significant decrease in the level of velocity and acceleration achieved with exercise was observed both in patients in Group I (normal thallium study) (1.2 +/- 0.3 m/s and 36.8 +/- 14 m/s per s, p less than or equal to 0.005) and in patients in Group II (ischemia) (1.1 +/- 0.3 m/s and 27.7 +/- 11 m/s per s, p less than or equal to 0.0005) compared with values in young control subjects (1.4 +/- 0.2 m/s and 52.7 +/- 16 m/s per s). When groups of patients of similar age who differed in the presence (Group II) or absence (Group I) of ischemia on thallium scintigraphy were compared, no difference was found for maximal velocity (1.1 +/- 0.3 versus 1.2 +/- 0.3 m/s, p = NS), but acceleration was significantly lower in Group II (27.7 +/- 11 versus 36.8 +/- 14 m/s per s, p less than or equal to 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of heart rate on Doppler indexes of systolic function in humans.

Recent investigations have shown Doppler echocardiography to be useful in the noninvasive assessment of left ventricular systolic function. No data exist, however, regarding the influence of heart rate on Doppler measurements of aortic blood flow velocity and acceleration in humans. Thus, 12 normal volunteers underwent continuous wave Doppler ultrasound recording from the suprasternal notch at baseline and during progressive transesophageal atrial pacing at intervals of 10 beats/min between 90 and 140 beats/min while 100% atrial capture and 1:1 atrioventricular conduction were maintained. Subjects were studied both upright (n = 12) and supine (n = 10). With the subject upright at baseline (mean heart rate 77.8 +/- 10.6 beats/min), peak acceleration averaged to 16.8 +/- 3.4 m/s2, and peak modal velocity and flow velocity integral averaged 0.72 +/- 0.14 m/s and 8.4 +/- 2.1 cm, respectively. With pacing at 90 beats/min, peak acceleration decreased to 15.6 +/- 3.6 m/s2, a significant decline from baseline values (p less than 0.005). Similar declines were seen during pacing at 90 beats/min for peak modal velocity and flow velocity integral (0.64 +/- 0.16 m/s and 7.1 +/- 1.9 cm, respectively; both p less than 0.005 versus baseline values). At the peak pacing rate of 140 beats/min, average peak acceleration decreased to 12.8 +/- 3.1 m/s2, and peak modal velocity and flow velocity integral decreased to 0.52 +/- 0.11 m/s and 5.02 +/- 1.25 cm, respectively. A significant linear correlation (r greater than or equal to 0.97, p less than 0.0001) was obtained for the relation between heart rate and peak acceleration, peak modal velocity and flow velocity integral.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative assessment of the hemodynamic consequences of aortic regurgitation by means of continuous wave Doppler recordings.

The purpose of this study was to evaluate the ability of continuous wave Doppler ultrasound recordings to reflect the magnitude and hemodynamic effects of aortic regurgitation. Forty-five patients with angiographically proved aortic regurgitation had Doppler studies performed within 24 hours of cardiac catheterization. High quality spectral recordings of the regurgitant jet were obtained in 31 patients, whereas 14 patients exhibited dropout of high velocity signals precluding measurement of maximal velocities. The slope of the peak to end-diastolic velocity decrease measured by Doppler examination was compared with the decay in the aortic to left ventricular diastolic pressure gradient by catheterization and was found to correlate well (r = 0.86). The Doppler velocity decay slope was generally higher in patients with angiographically severe rather than mild or moderate aortic regurgitation, but considerable overlap was present among groups. However, a diastolic velocity decay slope of greater than 3 m/s2 was seen only in those patients with advanced (3 or 4+) aortic regurgitation. Left ventricular end-diastolic pressure was estimated from the Doppler recordings by subtracting the end-diastolic pressure gradient obtained by the modified Bernoulli equation from the cuff diastolic blood pressure. A correlation was observed (r = 0.84) between Doppler and catheterization left ventricular end-diastolic pressure in the 31 patients with high quality spectral data, although the SEE was substantial (5.5 mm Hg). These data demonstrate that continuous wave Doppler recordings of the regurgitant jet can be useful in assessing the angiographic severity and hemodynamics of aortic regurgitation.

Influence of alterations in loading produced by lower body negative pressure on aortic blood flow acceleration.

The objectives of this study were to evaluate the effects of alterations in loading induced by lower body negative pressure on aortic blood flow velocity and acceleration. Twenty-seven normal men were studied during various levels of lower body negative pressure (0 to -60 mm Hg) during which echocardiographic, Doppler and hormonal measurements were obtained. Lower body negative pressure induced a decrease in left ventricular diastolic diameter from 5.18 +/- 0.08 to 4.41 +/- 0.1 cm (p less than 0.0001) and in left ventricular systolic diameter from 3.33 +/- 0.09 to 2.84 +/- 0.1 cm (p less than 0.0001). Shortening fraction remained unchanged. The decrease in diastolic diameter resulted in a reduction in flow velocity integral from 13.8 +/- 0.8 to 7.5 +/- 0.4 cm (p less than 0.0001) and, therefore, in stroke volume from 89.6 +/- 4.7 to 49.5 +/- 2.8 ml (p less than 0.0001). Heart rate reflexly increased from 62.5 +/- 1.9 to 82.2 +/- 2.3 beats/min (p less than 0.0001) as did systemic vascular resistance from 1,280.8 +/- 69.5 to 1,863.4 +/- 121.4 dyne.s.cm-5 (p less than 0.0001). The increase in heart rate was insufficient to maintain cardiac output, which decreased from 5.53 +/- 0.29 to 3.99 +/- 0.21 liters/min (p less than 0.0001). Systolic, diastolic and mean arterial blood pressure was maintained. The negative pressure resulted in a concomitant significant increase in norepinephrine levels from 1.46 +/- 0.09 to 2.056 +/- 0.2 nmol/liter (p = 0.0019) but no change in plasma epinephrine: 0.845 +/- 0.22 to 0.78 +/- 0.11 nmol/liter (p = NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Use of exercise Doppler echocardiography to evaluate cardiac drugs: effects of propranolol and verapamil on aortic blood flow velocity and acceleration.

This study evaluated the ability of exercise Doppler echocardiography to identify hemodynamic changes due to cardiac medication. Twenty young healthy volunteers (mean age 30 years) underwent continuous wave Doppler examination from the suprasternal notch at rest, during each stage of a standard exercise protocol and immediately after exercise. On completion of the control test, each subject received either 60 to 80 mg of propranolol or 120 mg of verapamil orally, and the same exercise protocol was repeated after 90 min. During the control test, values for modal velocity, acceleration and flow velocity integral all increased significantly from baseline (p less than 0.0002 for each). When exercise was repeated after propranolol administration, values for all Doppler measurements were significantly altered. Modal velocity at baseline was significantly lower after propranolol when compared with control (0.53 +/- 0.11 versus 0.63 +/- 0.17 m/s; p less than 0.0001). Similarly, modal velocity at maximal exercise was significantly lower after propranolol (1.11 +/- 0.2 versus 1.25 +/- 0.21 m/s; p less than 0.0001). The effect of propranolol on acceleration was even greater, with blunting of baseline (11.4 +/- 2 versus 15.4 +/- 5 m/s per s; p less than 0.0005) and exertional (33.4 +/- 10 versus 56.3 +/- 15 m/s per s; p less than 0.0001) acceleration. The flow velocity integral during exercise was greater after propranolol (14.1 +/- 3.1 versus 10.1 +/- 3.2 cm; p less than 0.0005) than during the control test. Verapamil failed to influence any Doppler-measured index of aortic blood flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Value and limitations of transesophageal echocardiography in determination of left ventricular volumes and ejection fraction.

Several formulas exist for estimating left ventricular volumes and ejection fraction using conventional two-dimensional echocardiography from transthoracic views. Transesophageal imaging provides superior resolution of endocardial borders but employs slightly different scan planes. The estimation of left ventricular volumes by transesophageal echocardiography has not been validated in human patients. Therefore, the purpose of this study was to compare left ventricular volumes and ejection fraction derived from transesophageal short-axis and four-chamber images with similar variables obtained from ventriculography. End-diastolic and end-systolic volumes and ejection fraction were calculated using modified Simpson's rule, area-length and diameter-length models in 36 patients undergoing left ventriculography. Measurements of left ventricular length were obtained from the transesophageal four-chamber view and areas and diameters were taken from short-axis scans at the mitral valve, papillary muscle and apex levels. Data from transesophageal echocardiographic calculations were compared with end-diastolic volume (mean 172 +/- 90 ml), end-systolic volume (mean 91 +/- 74 ml) and ejection fraction (mean 52 +/- 15%) from cineventriculography using linear regression analysis. The area-length method (r = 0.88) resulted in a slightly better correlation with left ventricular end-diastolic volume than did Simpson's rule (r = 0.85) or area-length (r = 0.84) formulas. For end-systolic volume, the three models yielded similar correlations: Simpson's rule (r = 0.94), area-length (r = 0.93) and diameter-length (r = 0.95). Each of the methods resulted in significant underestimation of diastolic and systolic volumes compared with values assessed with angiography (p less than 0.003). Ejection fraction was best predicted by using the Simpson's rule formula (r = 0.85) in comparison with area-length (r = 0.80) or diameter-length (r = 0.73) formulas. Measurements of left ventricular length by transesophageal echocardiography were smaller for systole (mean 5.7 +/- 1.6 cm) and diastole (mean 7.7 +/- 1.2 cm) than values by ventriculography (mean 9.2 +/- 1.4 and 8.1 +/- 1.6 cm, respectively; p less than 0.0001), suggesting that underestimation of the ventricular length is a major factor contributing to the smaller volumes obtained by transesophageal echocardiography. In conclusion, currently existing formulas can be applied to transesophageal images for predicting left ventricular volumes and ejection fraction. However, volumes obtained by these models are significantly smaller than those obtained with angiography, possibly because of foreshortening in the transesophageal four-chamber view.

Improved reproducibility of left atrial and left ventricular measurements by guided three-dimensional echocardiography.

OBJECTIVES: The objective of this study was to determine whether guided three-dimensional echocardiography could improve the reproducibility of left atrial and left ventricular anteroposterior measurements over that of standard unguided two-dimensional echocardiography. BACKGROUND: Although these measurements are standard indexes for evaluating chamber size, their use is limited by significant interobserver variability largely due to variable image plane positioning. To improve measurement accuracy and reproducibility, we have developed a three-dimensional echocardiograph that displays the line of intersection of the real-time image with a previously saved orthogonal reference image. This display shows the relation of the real-time image to anatomic landmarks in its third, nonvisualized dimension and may be used to guide image positioning. METHODS: Three pairs of operators independently performed unguided two-dimensional and guided three-dimensional examinations on three groups of 10 patients each. The left atrium was measured in a plane through the inferior surface of the aortic cusps and the left ventricle in a plane perpendicular to its long axis 1 cm below the mitral leaflet tips. Interobserver variability of these measurements on unguided parasternal long-axis images and on guided short-axis images was assessed. RESULTS: The standard unguided two-dimensional examination was associated with an interobserver variability of 14.6% and 9.1% for atrial and ventricular measurements, respectively. Guided three-dimensional echocardiography significantly reduced interobserver variability to 5.0% and 3.1%, respectively, for the same measurements (p < 0.005 by McNemar's test). CONCLUSIONS: Significant interobserver variability occurs with standard unguided two-dimensional echocardiographic measurement of left atrial and left ventricular dimensions. Guided three-dimensional echocardiography achieves a nearly threefold improvement of reproducibility of these measurements and provides the basis for improved serial evaluation and comparison of atrial and ventricular size by different operators.

Role of CD95/Fas and its ligand in the regulation of the growth of human CD34(++)CD38(-) fetal liver cells.

The functional significance of CD95/Fas expressed by candidate hematopoietic stem cells (HSCs) from human fetal liver was studied by testing the effect of agonistic anti-CD95 monoclonal antibody (mAb) CH-11 and soluble CD95 ligand (sCD95L) on the growth of CD34(++)CD38(-)lineage cells in vitro. Candidate fetal HSCs exhibited a dose-dependent proliferative response to CH-11 as well as to sCD95L when combined with kit ligand (KL) + interleukin 3 (IL-3) under serum-deprived culture conditions. CH-11 mAb increased, in a synergistic fashion, the number of myeloid colony-forming unit culture (CFU-C) generated by candidate HSCs in liquid cultures with the cytokine combinations KL + IL-3, KL + granulocytemacrophage colony-stimulating factor, and KL + IL-6. CH-11 mAb and sCD95L also enhanced erythropoiesis supported by KL + IL-3 + erythropoietin (Epo). Furthermore, sCD95L was able to increase the number of megakaryocytes, granulocytes, and CD34- cells generated in the presence of KL + IL-3 + Epo + thrombopoietin. An analysis performed using Western blotting revealed that the membrane-bound CD95L (mCD95L) was expressed by both immature (total CD34+/++) and mature (CD34-) hematopoietic lin(-) FL cells. Among the CD34(++)lin(-)cells, both the freshly isolated CD38+ and CD38 subsets as well as CD95+ and CD95- cells constitutively expressed mCD95L, demonstrating that the CD95/CD95L system represents a paracrine and potentially autocrine regulator of early hematopoiesis. To study the role of the endogenously produced CD95L, we determined the effects of a neutralizing anti-CD95L NOK-1 on the growth of candidate HSCs. By blocking the endogenous CD95L with NOK-1 mAb, we observed an increase in CFU-C generated by candidate HSCs. We conclude that the endogenous CD95L has an inhibitory effect on fetal candidate HSCs, which can be blocked by sCD95L and CH-11 mAb.

Effect of erythropoietic stress on donor hematopoietic cell expression in chimeric rhesus monkeys transplanted in utero.

We have previously reported the successful development of hematopoietic chimerism after the in utero transplantation of fetal hematopoietic stem cells (HSC) in rhesus monkeys (Macaca mulatta). These animals exhibit sustained engraftment without immunosuppression or graft-versus-host disease (GVHD). To assess the functional response of the donor-derived erythropoietic population, we assayed the relative expression of donor and recipient hematopoietic progenitors in chimeric monkeys before and after anemic stress. Anemia in our chimeric animals resulted in increased erythropoietin (EPO) production comparable to controls. This was accompanied by changes in erythroid progenitor profiles, again similar to controls. Chimeric animals demonstrated normal reticulocytosis and reconstituted their hematocrit after hemorrhage at the same rate as controls. The donor-derived erythropoietic population exhibited normal responses to recipient regulatory signals and did not seem to expand at the expense of other hematopoietic lineages. Thus the proportions of engraftment for the myeloid and erythroid precursors in bone marrow and for blood lymphocytes remained stable. Our results demonstrate that the in utero transplantation of fetal HSC results in stable engraftment of donor erythropoietic progenitors, which appear to be fully integrated within the recipient's regulatory system. The abnormalities reported in the postnatal transplantation setting can then be attributed to immunologic reactions requiring conditioning myeloablative regimens. Fetal transplantation bypasses all these factors.