Co-Graft of Allogeneic Immune Regulatory Neural Stem Cells (NPC) and Pancreatic Islets Mediates Tolerance,while Inducing NPC-Derived Tumors in Mice

Background

Data available on the immunomodulatory properties of neural stem/precursor cells (NPC) support their possible use as modulators for immune-mediated process. The aim of this study was to define whether NPC administered in combination with pancreatic islets prevents rejection in a fully mismatched allograft model.

Methodology/Principal Finding

Diabetic Balb/c mice were co-transplanted under the kidney capsule with pancreatic islets and GFP⁺ NPC from fully mismatched C57BL/6 mice. The following 4 groups of recipients were used: mice receiving islets alone; mice receiving islets alone and treated with standard immunosuppression (IL-2Rα chain mAbs + FK506 + Rapamycin); mice receiving a mixed islet/NPC graft under the same kidney capsule (Co-NPC-Tx); mice receiving the islet graft under the left kidney capsule and the NPC graft under the right kidney capsule (NPC-Tx). Our results demonstrate that only the co-transplantation and co-localization of NPC and islets (Co-NPC-Tx) induce stable long-term graft function in the absence of immunosuppression. This condition is associated with an expansion of CD4⁺CD25⁺FoxP3⁺ T regulatory cells in the spleen. Unfortunately, stable graft function was accompanied by constant and reproducible development of NPC-derived cancer mainly sustained by insulin secretion.

Conclusion

These data demonstrate that the use of NPC in combination with islets prevents graft rejection in a fully mismatched model. However, the development of NPC-derived cancer raises serious doubts about the safety of using adult stem cells in combination with insulin-producing cells outside the original microenvironment.     

Diagnostic application of first trimester trophoblast sampling in 100 pregnancies

Summary The results of the diagnostic application of first trimester trophoblast sampling in 100 pregnancies are reported in detail. Further improvement of the method for routine, direct chromosome analysis resulted in a technique which proved to be fast, simple, and efficient. We found that short-term incubation of villi permits the application of many experimental methods, such as visualization of sister chromatid exchanges and bromodeoxyuridine (BrdU) incorporation. Fetal karyotyping was successful in each of the 96 pregnancies in which fetal material was obtained from a total of 98 fetuses. There were 42 males and 56 females, and an abnormal chromosome constitution was found in 12 cases. Two trisomic fetuses were found among the eight pregnancies at risk for Duchenne muscular dystrophy, and this indicates that fetal sexing (which is achieved with our method in two hours) should not be performed without chromosome visualization. The results indicate a risk of 8% of an abnormal fetus for mothers aged 35 years or more, while the risk of failure of sampling and of spontaneous abortion after villi sampling were 4 and 6%, respectively. Enzyme determinations were performed in three pregnancies at risk for gangliosidosis GM₁, Niemann-Pick disease, and Hurler syndrome. In this last case inconsistency between the results of the assay of iduronidase on chorionic villi and amniotic fluid cells was found. This unexplained error indicates the need for extensive characterisation in chorionic villi of the series of enzymes involved in metabolic diseases.     

Some genetic aspects of meiosis in males of Drosophila melanogaster,carriers of In(1)sc 4 sc 8

In order to verify the theory of non-homologous pairing in the male meiotic metaphase of Drosophila melanogaster, particular crosses were carried out with In(1)sc ⁴ sc ⁸ males, with or without the inversion In(2)SM ₅, Cy on the second chromosome. The results seem to indicate the existence of the above mechanisms, which would appear to contribute, not infrequently, to the overall non-disjunction of the In(1)sc ⁴ sc ⁸+Cy males.The work has been financially supported by the Consiglio Nazionale delle Ricerche (CNR).     

Effects of seminal plasma and three extenders on canine semen stored at 4 degrees C

Semen preservation and artificial insemination (AI) in the canine has become a common practice in veterinary medicine. Chilled dog semen is easy to handle, and several extenders can be used. The aim of this study was to compare the effects on canine spermatozoa of seminal plasma and 3 extenders commonly used for chilled semen preservation in clinical practice. The characteristics evaluated were sperm motility; velocity; plasma membrane status (assessed with a fluorescence staining technique and hypo-osmotic swelling test); acrosome morphology; semen pH; and semen osmolarity. These criteria were monitored daily in the ejaculates of 11 dogs. The ejaculates were divided into 4 aliquots. Each aliquot was extended in autologous seminal plasma, egg-yolk Tris, egg-yolk milk or egg-yolk cream and preserved at 4 degrees C for 4 d. In 10 of 11 semen samples extended in autologous seminal plasma, motility had already decreased to 0% by Day 2, and the percentage of spermatozoa with intact membranes was lower than in the 3 extenders (P < 0.05). Motility up to Day 4 was higher in egg-yolk Tris-stored spermatozoa (53.6%) than in those preserved in egg-yolk milk (30.4%) and egg-yolk cream (14.1%). Spermatozoa stored in egg-yolk Tris also had the highest sperm velocity, whereas no difference was found in plasma membrane or acrosome status (P>0.05). Egg-yolk Tris extender seems to be superior to the other extenders tested, to preserve dog semen at 4 degrees C, although differences were not significant for all the parameters.     

Immunocytochemical characterization of human NOR-90 (upstream binding factor) and associated antigens reactive with autoimmune sera

The 90-kDa nucleolus organizer region autoantigen (NOR-90) was previously shown to be identical to the human upstream binding factor (hUBF) and composed of twoMr forms. In this study, thirteen human anti-NOR-90/hUBF autoimmune sera were used to further characterize NOR-90/hUBF and its associated autoantigens. Nucleolar and nucleoplasmic staining of interphase cells and NOR staining in mitosis were observed with all sera by immunofluorescence. All sera showed equal reactivity with both high and lowMr forms in Western blotting and immunoprecipitation, suggesting that the cellular content and distribution for bothMr forms were approximately equal. Using extracts of [³⁵S]methionine- and [³²P]orthophosphate-labeled cells, phosphorylated and nonphosphorylated NOR-90/hUBF were identified for bothMr forms and these two populations were recognized by human autoantibodies. In immunoprecipitation analyses, the nonphosphorylated population was readily extracted while the phosphorylated population was tightly bound. Clinical data were available for 8 patients in whom anti-NOR-90/hUBF autoantibodies were present. They had diverse diagnoses including SLE, rheumatoid arthritis and malignancies. Although only one patient was diagnosed as scleroderma, Raynaud's phenomenon was observed in 4 of the 8 patients. Interestingly, one NOR-90/hUBF serum was shown to contain additional antibodies to RNA polymerases I and II.Abbreviations: ANA=Antinuclear antibody; HCC=hepatocellular carcinoma; hUBF=human upstream binding factor; NOR=nucleolus organizer region; RNA pol I=RNA polymerase I; RNA pol II=RNA polymerase II; rRNA=ribosomal RNA; SLE=systemic lupus erythematosus.Second Department of Internal Medicine, Shinshu University School of Medicine, Matsumoto-Shi 390, Japan     

A novel mechanism for the origin of supernumerary marker chromosomes

A ring chromosome 3 and a 47^th chromosome formed by the portions of 3p and 3q distal to the r(3) breakpoints were found in a girl with mental retardation and minor facial anomalies. The supernumerary chromosome 3, rea(3), had a primary constriction inside its 3p portion (3p23) and was consistently stable both in lymphocytes and fibroblasts. In situ hybridization with alphoid probes revealed that the r(3) maintained its wild-type centromere, whereas the rea(3) showed no alphoid-related signals. This case and a similar one recently reported demonstrate that acentric fragments can acquire a new centromere and become stable, and that supernumerary marker chromosomes can also originate by the junction of the acentric portions distal to the centric region forming a ring. The possibility of such a chromosome segregating will depend on its ability to (re)activate a new centromere.     

Genetic variation and natural hybridization betweenOrchis laxiflora andOrchis palustris (Orchidaceae)

Genetic divergence between population samples ofOrchis laxiflora and ofO. palustris from various European locations was studied by electrophoretic analysis of 25 enzyme loci. An average genetic distance of D_Nei = 1.24 was found between the two taxa, with 12 out of 25 loci showing alternative alleles (diagnostic loci). Genetic heterogeneity was observed within bothO. laxiflora andO. palustris, when northern and southeastern populations were compared, being lower in the former taxon (D = 0.06), than in the latter (D = 0.16). Karyologically, 2n = 36 was found for bothO. laxiflora andO. palustris. O. laxiflora andO. palustris produce hybrids, described asO. ×intermedia. Genotype analysis of several sympatric samples showed the presence of hybrid zones, including F₁ hybrids and, in low proportions, recombinant classes, putatively assigned to F_n and backcrosses, as well as a few introgressed individuals of both taxa. These data indicate that hybrids are only partially fertile, with a very limited mixing up of the two parental gene pools; this is also shown by the lack of significant lowering of genetic distances when sympatric and allopatric heterospecific samples are compared. Accordingly,O. laxiflora andO. palustris form a syngameon; nevertheless they can be considered as good taxonomic species, with virtually distinct gene pools, which evolve independently. The genetic variability inO. laxiflora andO. palustris is remarkably low ( _e = 0.05 and _e = 0.02, respectively). In particular, nearly complete absence of polymorphic loci was found inO. palustris from northcentral Europe. Two hypotheses are considered to explain the low genetic variability of this endangered species.     

Adhesion and activation molecules expressed by human natural killer cells

Our study concerns the expression and the regulation of adhesion and activation receptors on human NK cells. In particular we provide evidence on: a) the expression on fresh human NK cells of VLA-4, VLA-5 and VLA-6, extracellular matrix (ECM) receptors of integrin family capable of mediating their adhesion to FN and LM; b) the role of PKC on the regulation of CD16, a differentiation antigen associated with FcR type III expressed by all NK cells, which mediate ADCC activity and trigger lymphokine production.