Smoking during early pregnancy affects the expression pattern of both nicotinic and muscarinic acetylcholine receptors in human first trimester brainstem and cerebellum.

Prenatal nicotine exposure is associated with an increased risk of complications during pregnancy and childhood. In this study the expression of nicotinic and muscarinic acetylcholine receptors in first trimester pons, medulla oblongata and cerebellum from abortus (5-12 weeks of gestation) of smoking and nonsmoking women was compared. A significant age-related increase in binding of nicotinic receptor subtype alpha4 was found in both pons and cerebellum only in fetal tissue from non-smoking women, while a similar increase was observed in medulla oblongata from fetuses exposed to smoking. A significant age-related increase in binding of muscarinic receptor subtype m2 was observed in pons from abortus of smoking compared with non-smoking women. The gene expression pattern of both alpha4 and alpha7 nicotinic receptor subunits was changed after smoking in all three regions investigated. Smoking also changed the expression of m1 and 2 muscarinic receptor mRNA in pons, m1 mRNA in cerebellum and the m3 mRNA in medulla oblongata. The findings indicate that early prenatal nicotine exposure affects the normal developmental pattern of the cholinergic system in human fetal brain.

Distribution of presenilin 1 and 2 and their relation to Notch receptors and ligands in human embryonic/foetal central nervous system.

Notch signaling in vertebrates is mediated by four Notch receptors (Notch-1, -2, -3, and -4) that are activated by interacting with at least five different Notch ligands, Jagged-1, Jagged-2, Delta-1, -2, and -3. Recent studies have shown that the gamma-secretase-like intramembranous cleavage of Notch receptors to release their cytoplasmic signaling domains requires the presenilin (PS) proteins 1 and 2 (PS1 and PS2). Here, we used immunohistochemistry to compare the distribution of all four Notch receptor proteins and three ligands in the context of co-localization with PS1 and PS2 in first trimester human central nervous system (CNS). In addition, we investigated Notch receptors and ligands expression by Western blotting. The study was performed on the forebrain and spinal cord of human embryonic/foetal CNS (5-11 gestational weeks). Results showed a divergent distribution of the different Notch receptor proteins with only Notch-1 being co-localized with PS1 and PS2. Notch-2 was only seen occasionally within the developing cortex and spinal cord. Notch-3 expression was restricted to neuroepithelial cells of the spinal cord and endothelial cells in blood vessels of both developing cerebral cortex and spinal cord. The weak, punctate staining of Notch-4 in the neuroepithelium of the spinal cord could not be confirmed with Western blotting. Neither Notch-2, nor -3 showed overlap with either PS1 or PS2 immunoreactivity. The ligand Jagged-1 was found sporadically in the neuroepithelial cell layer in cerebral cortex of the earlier stages of development and of the spinal cord during the first trimester while Jagged-2 was not detected. Jagged-1 and Jagged-2 immunoreactivities were not found in the 9-11-week cortex. No co-distribution of Jagged-1 and PS1 or PS2 was found. Delta-1 ligand expression was detected in neuroepithelial cells of the ventricular zone of the cerebral cortex, and also in maturating neurons in the cortical plate and ventral horns of the developing spinal cord. The presence of Notch-1, Delta-1 and Jagged-1 in the neuroepithelium of developing CNS indicates that Notch signaling in proliferating human progenitor cells only involves these two receptor ligands and that cleavage of Notch-1 is mediated both by PS1 and PS2. The strong immunoreactivity of Notch-1, Delta-1 and PS1 in the cortical plate and in maturating neurons of the spinal cord also suggests that these proteins may regulate the maturation processes of post-mitotic neurons. The pronounced PS1 immunoreactivity in neurites in the hindbrain and spinal cord without detectable expression of any Notch receptor or ligand suggests that a possible role for PS1 in neurite growth involves either gamma-secretase-mediated cleavage of other substrates or gamma-secretase-independent mechanisms.

Nicotine-induced alterations in the expression of nicotinic receptors in primary cultures from human prenatal brain.

The nicotinic receptor proteins and gene transcripts for the different nicotinic receptor subunits exist in human prenatal brain already at 4-5 weeks of gestation. The early presence of nicotinic receptors suggests an important role for these receptors in modulating dendritic outgrowth, establishment of neuronal connections and synaptogenesis during development. When measurements of nicotinic receptors using [(3)H]epibatidine (labelling both the alpha3 and alpha4 subtype) and [(3)H]cytisine (labelling the alpha4 subtype) were performed in intact cells from the cortex, subcortical forebrain and mesencephalon (7.5-11 weeks of gestation), the highest specific binding for both ligands was detected in cells from mesencephalon, followed by subcortical forebrain and cortex. The effects of nicotine exposure were studied in primary cultures of prenatal brain (7.5-11 weeks of gestation). Treatment with nicotine (1-100 microM) for 3 days significantly increased the specific binding of [(3)H]epibatidine and [(3)H]cytisine in cortical cells but not in cells from subcortical forebrain and mesencephalon brain regions, indicating region-specific differences in the sensitivity to nicotine exposure. Relative quantification of mRNA showed that the expression of the nicotinic receptor subunits alpha3 and alpha7, but not alpha4, was increased in cortical cells after nicotine treatment. These findings support the assumption of a potential risk of disturbance in the functional role of nicotinic receptors during brain development as a consequence of maternal smoking during pregnancy.

Solid human embryonic spinal cord xenografts in acute and chronic spinal cord cavities: a morphological and functional study.

While therapeutic spinal cord grafting procedures are of interest in the chronic spinal cord injury stage, previous experimental grafting studies, including human spinal cord tissue, have mainly focused on the acute stage. Therefore, solid human embryonic spinal cord grafts were implanted in acute or chronic spinal cord aspiration cavities of immunodeficient rats to compare the morphological and locomotor outcome to that of lesion alone cases. Locomotor function was assessed using the Basso, Beattie, and Bresnahan open-field locomotor rating scale up to 6 months, while the morphological evaluation of graft survival, growth, and integration was performed at 6 weeks or 6 months after implantation. Graft survival was 94% in both lesion models, while graft growth was enhanced in the chronic compared to the acute cavity group. Human specific Thy-1 and neurofilament immunoreactive fibers were observed up to 7 mm into host white matter, while aminergic fibers were observed up to 1 mm into the grafts. Abundant calcitonin gene-related peptide immunoreactive fibers in the grafts in the absence both of immunoreactive cell bodies and colocalized human-specific neurofilament immunoreactivity, suggested host fiber ingrowth. At 6 months, the grafted cases presented less central canal deformation and lower glial fibrillary acidic protein immunoreactivity at the host cavity border compared to that of the nongrafted cases. The strong compensatory regain of locomotor function after unilateral spinal cord lesions was not affected by the human spinal cord grafts. In conclusion, solid human embryonic spinal cord tissue transplanted to a cavity in the adult injured spinal cord results in beneficial morphological effects in both the acute and chronic spinal cord lesion.

Expression of presenilin-1 and Notch-1 receptor in human embryonic CNS.

In vitro studies have shown that the Alzheimer's disease-related presenilin-1 protein can mediate Notch-1 receptor cleavage during signalling. In the present study, we compared the distribution of presenilin-1 and Notch-1 receptor immunoreactivities in human embryonic CNS tissue during the first trimester of development. Our aim was to gain insight into whether these proteins are likely to interact functionally during human fetal brain development. CNS material was obtained from routine abortions, cryosectioned and studied by means of immunohistochemistry with antibodies to presenilin-1 and Notch-1. At very early stages of embryonic development (four to five gestational weeks) intensive presenilin-1 immunoreactivity could be seen predominantly in neurites in the ventral horn of the spinal cord, where it overlapped with 200-kDa neurofilament immunoreactivity. Presenilin-1 immunoreactivity was also seen in neuroblasts of the ventricular zone of the tel- and mesencephalon, as well as of the brainstem. Notch-1 receptor appeared in neuronal and ependymal cells throughout the CNS. Seven- to eight-week CNS tissue showed similar patterns of presenilin-1 and Notch-1 receptor expression in the spinal cord and cerebral cortex as was seen at five weeks. Both proteins were localised in the neuroepithelial cell layer lining the ventricles, as well as in the cortical plate layer, where immunoreactivity was seen in the cell bodies. In addition, presenilin-1 immunoreactivity was seen in thin neurites in the subplate of the developing cortex. At 10 weeks, presenilin-1 immunoreactivity was reduced in the spinal cord. These results show that, although presenilin-1 and Notch-1 receptor are localised to the same differentiating cell populations in the human cerebral cortex, making a direct interaction possible, these proteins are otherwise confined to different neurons or neuronal compartments, suggesting a role for presenilin-1 during early CNS differentiation that does not involve Notch-1 receptor processing. Double staining for presenilin-1 in the endoplasmic reticulum and presenilin-1 in the Golgi showed overlap to some extent in investigated CNS regions, but not in neurites. This suggests that presenilin-1 function during neurogenesis is not exclusively correlated to protein processing within the endoplasmic reticulum and Golgi, but that presenilin-1 may also be involved in other processes, such as axonal and dendritic outgrowth or synaptic formation. In summary, our findings provide supportive evidence that the presenilin-1 protein is involved in the development and maturation of the human fetal CNS. The presence of presenilin-1 immunoreactivity in both the cell bodies and neurites of developing neurons strongly suggests divergent mechanisms of function for presenilin-1 during human brain development. These may include interactions with any of the Notch receptor proteins, as well as Notch-independent mechanisms.

MHC antigen expression in human first trimester spinal cord with implications for clinical transplantation procedures.

We report human leukocyte antigen (HLA) class I expression in 5-17% and class II in 0-9% of first trimester human spinal cord cells. After 8 days in culture with gamma-interferon, >87% of the spinal cord cells expressed HLA class II. However, mixed cultures of adult human peripheral lymphocytes and immature human spinal cord cells, showed no induction of lymphocyte proliferation prior to or after gamma-interferon exposure in culture. In conclusion, we report non-immunogenic expression of HLA antigens in the human first trimester spinal cord.

Mixed lymphocyte culture of human fetal liver cells.

OBJECTIVE: In order to study the immunological function of the human fetus in the first and second trimesters, mixed lymphocyte culture (MLC) of fetal liver and thymic cells was performed. MLC is a functional test to determine human lymphocyte antigen-D incompatibilities. METHODS: Human fetal liver and thymic tissue was obtained from abortions in gestational weeks 7-17.5. Forty-seven fetuses were studied with one-way MLC. The cells were stimulated by adding irradiated fetal liver cells, adult bone marrow and peripheral blood lymphocytes. The activity was measured as DNA incorporation of radiolabeled thymidine. RESULTS: The results indicate that the human fetus is competent to react as early as 11-12 weeks of gestation and in some cases even earlier. In very immature fetal livers (< 8 weeks), the MLC seems to be inhibited. CONCLUSIONS: Our data suggest that the human fetus can react against foreign transplantation antigens earlier than previous papers have claimed. The onset of reactivity seems to differ considerably among fetuses. The present findings may explain some of the limited success of in utero transplantations of hematopoietic stem cells in human fetuses of normal immunological status.

Ionotropic glutamate receptor expression in human spinal cord during first trimester development.

Quantitative receptor autoradiography and immunoblotting were used to study the expression and distribution of AMPA, kainate and NMDA receptors in first trimester human spinal cord obtained from elective abortions ranging from 4 to 11.5 weeks of gestational age. Spinal cord tissue sections were processed for receptor autoradiography with the ligands [3H]AMPA, [3H]kainate and [3H]MK-801 and the optical density was measured separately in a dorsal region (alar plate) and ventral region (basal plate) of the autoradiographs. Binding sites for all three ligands were demonstrated already at 4-5.5 weeks of gestation and increased continuously during the first trimester both in the dorsal and ventral regions. [3H]AMPA binding to both high- and low-affinity sites increased from undetectable levels to about 35 and 400 fmol/mg tissue, respectively, during this period. A temporal difference in the distribution of [3H]AMPA binding sites was observed. The early homogeneous pattern of [3H]AMPA binding in both alar and basal plates had changed to a heterogeneous pattern at 11 weeks of gestation with the highest density of [3H]AMPA binding sites in the superficial layers of the immature dorsal horn. [3H]kainate and [3H]MK-801 binding sites were densely and homogeneously distributed already at 4 weeks, and steadily increased six- and two-fold, respectively, to about 100 fmol/mg tissue at 11.5 weeks of gestation. Immunoreactive bands corresponding to the NMDA receptor subunits NR1, NR2A, NR2B, NR2C and NR2D were demonstrated by immunoblotting at the earliest between 4.5 and 7 weeks and increasing concentrations were seen up to 11 weeks of gestation. These results suggest that AMPA, kainate and NMDA receptors are expressed in the human spinal cord early in embryogenesis.

Human first trimester forebrain cells express genes for inflammatory and anti-inflammatory cytokines.

Using in situ hybridization and immunohistochemistry the induction of potential cytokines [interleukin 1beta (IL-1beta), IL-4, IL-6, IL-10, tumour necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma) and transforming growth factor beta (TGF-beta)] in human embryonic forebrain cells at weeks 5, 7 and 10 of gestation was studied. The aims were to investigate the capacity of these cells to express cytokines, to determine the kinetics of induction and to display the type of cytokine expressing cells in the developing brain. Constitutive cytokine gene expression was recorded from week 5, augmented by about 50% at week 7 and by more than 100% at week 19 (except TGF-beta at week 10). Among other cytokines, IL-1beta exhibited the highest expression at week 10. TGF-beta showed maximum expression at week 7 and declined at week 10. Combined techniques revealed that glial cells are a major source of cytokines. The study show and present for the first time the kinetics of spontaneous cytokine expression in human embryonic forebrain cells. The increased mRNA expressions with age suggest an important role for cytokines in promotion of brain development. The capacity of inducing these cytokines may, however, be implicated in the immunopathogenesis of several brain diseases. The distinctive TGF-beta profile suggests a further role for TGF-beta on modulation of cytokine responses during development.

Interactions between human embryonic forebrain cells and the cytokines interferon-gamma and interleukin-4.

In the present study we investigated the capacity of human first trimester forebrain cells at different gestational ages to produce IFN-gamma and IL-4. We also studied the effects of IFN-gamma on their proliferation, survival and expression of MHC antigens. IFN-gamma but not IL-4 was spontaneously produced after 24 h cultures. Furthermore, IFN-gamma exhibited anti-proliferative effects and induced MHC expression on these cells. However, the IFN-gamma exposed cultures showed significantly higher cell survival compared to un-exposed cultures. Co-culture with IL-4 blocked the IFN-gamma production and reversed its anti-proliferative effects. These interactions suggest important roles for cytokines in the survival, proliferation and differentiation of human embryonic and fetal forebrain cells.

Hydatidiform mole with non-metastatic pulmonary complications and a false low level of hCG.

We present a case of hydatidiform mole with non-metastatic pulmonary complications and stress that termination of the pregnancy will cure the patient. The frequent use of sonography in early pregnancy makes it possible to diagnose pathological pregnancies earlier than was possible before. The fact that molar pregnancies are now being terminated at an earlier stage means that some of the complications associated with advanced moles are seldom encountered today. The case also illustrates the so-called 'high dose hook effect' meaning that an extremely high level of hCG may falsely be reported to be very low by the laboratory.

Human embryonic spinal cord grafts in adult rat spinal cord cavities: survival, growth, and interactions with the host.

The ability of solid pieces of transplanted human embryonic spinal cord to survive, grow, and integrate with adult rat host spinal cord tissue was investigated. Unilateral cavities were surgically created at vertebral level T12-T13 in 10 athymic nude rats and 5 regular Sprague-Dawley rats. Seven of the athymic rats acutely received a human spinal cord graft, while the remaining 8 rats served as controls, with cavities alone. After 6 months the morphological outcome was evaluated with cresyl violet and with immunohistochemistry using antibodies toward human-specific neurofilament (hNF), human-specific Thy-1 (Thy-1), neurofilament, glial fibrillary acidic protein, serotonin (5-HT), and tyrosine hydroxylase (TH). The in situ morphology of the human embryonic spinal cord was also investigated and compared with grafts that were six months older. Solid human embryonic spinal cord grafts showed a 100% survival rate, grew to fill the volume of the cavity in a noninvasive manner, and expressed human specific antigens 6 months postgrafting. Thy-1 immunoreactivity (IR) was demonstrated up to 8 mm rostral to the graft suggestive of graft-derived fiber outgrowth. hNF-IR fibers and 5-HT- and TH-IR fibers traversed the graft-host border for a few hundred micrometers, respectively. Finally, our findings suggest that grafted solid pieces of human embryonic spinal cord minimize cystic deformations seen in the adult rat spinal cord with a unilateral cavity.

Tissue distribution of transplanted fetal liver cells in the human fetal recipient.

OBJECTIVE: Our purpose was to study the tissue distribution and concentrations of transplanted fetal liver cells in the human fetus. STUDY DESIGN: Radiolabeled indium 111 fetal liver cells were injected in vivo under ultrasonographic guidance into 10 normal fetuses (13 to 17 weeks of gestation) before a prostaglandin abortion. Six fetuses were injected intraperitoneally and four intracardially. Another two fetuses serving as controls were injected with indium-labeled maternal plasma. The fetuses were all alive, at least until 6 hours before expulsion. After expulsion the fetuses were dissected, and radioactivity was measured in various fetal tissues. Results for each tissue were expressed as percentages of the total injected dose. RESULTS: Significantly greater uptake of fetal liver cells in the liver, spleen, thymus, kidney, lung, and placenta was obtained with intracardiac than with intraperitoneal injection. Skeletal uptake did not differ in relation to mode of administration. With intracardiac injection uptake was greater in such parenchymal organs as the liver, spleen, and thymus (4.9%, 4.0%, and 3.9%, respectively). Uptake in the rib, clavicle, humerus, and sternum was 2.7%, 1.8%, 2.1%, and 1.1%, respectively. Placental uptake was 0.1%. The intracardiac route yielded a higher concentration of cells in different fetal organs than did injection of only radiolabeled maternal plasma, suggesting an active uptake of cells in different fetal hematopoietic organs. CONCLUSION: The mode of administration of fetal liver cells seems to be a major determinant of donor cell concentration in the transplanted human fetus and may be a significant determinant of the rate of successful engraftment.

Obliteration of a posttraumatic spinal cord cyst with solid human embryonic spinal cord grafts: first clinical attempt.

Cystic lesions of the spinal cord (syringomyelia) may occur after spinal cord injury. Posttraumatic syringomyelia may result in a myelopathy causing symptoms of sensory and motor loss, as well as worsening spasticity, pain, hyperhidrosis, and autonomic dysreflexia. Shunting of the cyst cavity along with untethering of the scarred spinal cord is widely accepted as the treatment of choice. However, the long-term stabilization of the progressive myelopathy caused by a posttraumatic cyst is suboptimal because of arachnoidal rescarring, shunt tube blockage, and cyst reexpansion. A new neurosurgical strategy to overcome the complication of cyst reexpansion was designed. Experimental studies have shown the successful use of embryonic spinal cord grafts, including human grafts, to obliterate induced spinal cord cavities in rats. The authors report the first use of solid human embryonic spinal cord grafts to successfully obliterate 6 cm of a large cyst cavity in a patient becoming myelopathic from a posttraumatic cyst. The grafts are well visualized by MRI to the 7-month postoperative follow-up and cyst obliteration is seen in the region where the grafts were placed.

Lack of evidence of permanent engraftment after in utero fetal stem cell transplantation in congenital hemoglobinopathies.

The use of fetal hematopoietic stem cells for in utero transplantation to create permanent hematochimerism represents a new concept in fetal therapy. In one fetus with alpha-thalassemia, one with sickle cell anemia, and one with beta-thalassemia, we have transplanted fetal liver cells obtained from legal abortions in gestational weeks 6-11. The fetus with alpha-thalassemia was transplanted twice during pregnancy, in the 15th (20.4 x 10(8) cells/kg) and in the 31st weeks of gestation (1.2 x 10(8) cells/kg), and is now two years of age. One fetus with sickle cell anemia received its transplant in the 13th week of gestation (16.7 x 10(8) cells/kg), and is now one year old. The fetus with beta-thalassemia was transplanted in 18th week (8.6 x 10(8) cells/kg), and is now three months old. Engraftment was evaluated by chromosomal analysis (sex chromosomes), red cell phenotyping, HLA class I and II typing, and PCR (polymerase chain reaction) for Y chromosome-specific sequences and DNA polymorphisms in cord and peripheral blood. The children with alpha- and beta-thalassemia underwent bone marrow aspirations at 3 and 7 months of age, respectively. In neither of these cases were we able to detect convincing evidence of stem cell engraftment. Thus, the administration of fetal stem cells to fetal recipients after the 12th week of gestation did not result in permanent hematochimerism. It remains to be determined whether the engraftment process can be promoted by earlier transplantations and/or higher cell doses.

Increased release of tissue plasminogen activator and its inhibitor in human parotid saliva upon stimulation.

Parotid saliva from 12 healthy volunteers was collected prior to and after 5 and 25 min of stimulation at a constant flow rate of 0.25 or 1.0 ml min-1. In the salivary samples the concentrations of tPA (tissue-type plasminogen activator), PAI-1 (plasminogen activator inhibitor type-1), albumin and total protein were determined and the activity of amylase, tPA and PAI assessed. Presence of both tPA and PAI-1 antigen was demonstrated in all samples, and in unstimulated saliva the ratio between the activator and its inhibitor was 1:7. Upon stimulation we found a significantly increased concentration of PAI-1, a less pronounced increase in tPA concentration, unchanged amylase and total protein levels and significantly decreased albumin concentration. tPA activity was significantly reduced after prolonged stimulation which had no effect on PAI activity. In stimulated saliva a significant positive correlation between concentration of tPA and PAI-1 was demonstrated. Stimulation with citric acid had no effect on output of albumin which is passively filtered from blood, whereas the increase in flow rate corresponded to the significantly increased secretion rate of total protein and amylase which is secreted by gland cells. The secretion pattern of tPA and PAI-1 differed significantly from that of albumin in showing markedly increased output rate during the stimulation period, and the relative increase in output of PAI-1 was significantly higher than that of amylase and total protein. Thus, the results from this study suggest an active release of both tPA and its main inhibitor PAI-1 into saliva.

Haematocervix after conization diagnosed by ultrasonography.

Haematocervix is an uncommon complication after conization. This report deals with 1 case that presented with clinical signs of progressive stenosis and was diagnosed by ultrasonography. Based on analysis of the 3 previously reported cases the pathogenesis of this condition is discussed.

Presence of a fast-acting specific inhibitor of plasminogen activator in human parotid saliva.

The plasminogen activator in parotid saliva has recently been characterized as a tissue-type plasminogen activator (t-PA). In this study stimulated parotid saliva from 21 healthy volunteers was analysed for (a) t-PA activity using the fibrin plate assay or a bioimmunoassay coupled to the plasmin-specific chromogenic substrate S-2251, (b) t-PA antigen using an enzyme-linked immunospecific assay and (c) activity of the specific fast-acting plasminogen activator inhibitor (t-PA inhibitor) assayed by its inhibitory effect on one-chain t-PA added to the samples. In parotid saliva the median t-PA activity was 0.2 IU ml-1 (normal range 0.05-0.35 IU ml-1), but activity of free t-PA could not be demonstrated by bioimmunoassay in any sample. The mean antigen concentration of t-PA was 2.2 IU ml-1 in the 10 samples with levels above the detection limit (0.5 IU ml-1). High activity of t-PA inhibitor was demonstrated in all parotid salivas, and the mean inhibitory effect on t-PA was 13.2 IU t-PA quenched per millilitre. This study thus demonstrates a fibrinolytic system in parotid saliva characterized by high t-PA inhibitor activity and relatively low concentration of inactive, probably complex-bound, t-PA which is activated in the presence of fibrin.

Effect of flow rate on tissue plasminogen activator activity in human parotid saliva.

Parotid saliva from 10 healthy volunteers was collected at rest and at constant flow rates of 0.25, 0.5 and 1.0 ml/min, and its tissue plasminogen activator (tPA) activity assayed on fibrin plates containing plasminogen. In unstimulated salivas the median tPA activity was 0.26 (range 0.03-2.0) IU/ml. During the first 15 min of stimulation, a continuous decrease in tPA activity was found at the three flow rates; thereafter a steady state was obtained. No significant differences in activity were found between the three rates. The initial decrease was on average 0.15 IU/ml, and the activity during the steady state was 38% of the prestimulatory level. Thus, stimulation with citric acid causes a significant decrease in tPA activity of parotid saliva but this decrease, as well as the reduction rate, appears to be independent of flow rate.