Human Fetal Testicular Tissue Xenotransplantation: A Platform to Study the Effect of Gonadotropins on Human Germ Cell Development In Utero.

PURPOSE: We examined the effects of long-term hCG stimulation on germ cell maturation, and Sertoli and Leydig cell function in a xenotransplantation model of the human fetal testis. MATERIALS AND METHODS: A total of 20 human fetal testes were ectopically xenografted on 20 castrated NCr male nude mice. Grafts were collected for analysis 24 weeks later. Mice were treated with saline as the control or with hCG beginning 4 weeks after the grafts were transplanted. RESULTS: Of the grafts 65% survived at 24 weeks. In contrast to untreated pregrafted samples, hCG stimulated xenografts showed significantly increased density of seminiferous tubule formation with Sertoli cell migration to the basement membrane. Germ cell proliferation and differentiation from gonocytes (M2A(+)) to prespermatogonia (MAGE-4A(+)) were observed in graft samples recovered from the hCG and nonhCG treated groups at 24 weeks of treatment. Leydig cells in hCG treated grafts produced significantly more testosterone than nonhCG treated grafts. Although further studies are required to investigate the potential for further differentiation and maturation of xenografted human fetal testes, normal in utero testicular development was reproduced under long-term hCG stimulation. CONCLUSIONS: This model represents a means to study long-term effects of gonadotoxins or hormonal stimulation on the maturation of human fetal testes.

The fibrinogen but not the Factor VIII content of transfused plasma determines its effectiveness at reducing bleeding in coagulopathic mice.

BACKGROUND: The evidence supporting plasma transfusion as a means to restore hemostatic control and prevent or treat bleeding is weak, leading to uncertainties as to which proteins affect the therapeutic quality of plasma. Some regulators focus on coagulation Factor (F)VIII activity, but whether this measure reflects overall transfusable plasma efficacy is questionable. We developed a mouse model of coagulopathy in which bleeding outcomes were responsive to plasma transfusion and addressed the relative contributions of FVIII and fibrinogen (Fg) to plasma quality. STUDY DESIGN AND METHODS: Anesthetized mice were rendered coagulopathic by four rounds of exchange of whole blood for washed red blood cells (RBCs) in 5% human albumin solution (HAS), which reduced RBCs, platelets, and plasma protein levels by 55, 66, and 80% of starting levels, in a blood exchange-induced coagulopathy approach (BECA). Before tail vein transection, BECA mice were transfused with HAS, wild-type murine fresh-frozen plasma (WT mFFP), or mFFP from FVIII-/- or Fg-/- knockout mice. BECA mice were also subjected to laser-induced arteriolar injury and thrombus formation quantified by intravital microscopy. RESULTS: Transfusion of WT or FVIII-/- mFFP reduced blood loss by fourfold in BECA mice relative to HAS; Fg-/- mFFP had no effect. WT or FVIII-/- mFFP transfusion, but not that of Fg-/- mFFP, increased thrombus size in laser-injured BECA mice arterioles. Extended refrigerated storage of mFFP did not reduce its antihemorrhagic effects. CONCLUSIONS: The content of Fg, but not FVIII, determined the efficacy of plasma transfusion in coagulopathic mice.

The complete N-terminal extension of heparin cofactor II is required for maximal effectiveness as a thrombin exosite 1 ligand.

BACKGROUND: Heparin cofactor II (HCII) is a circulating protease inhibitor, one which contains an N-terminal acidic extension (HCII 1-75) unique within the serpin superfamily. Deletion of HCII 1-75 greatly reduces the ability of glycosaminoglycans (GAGs) to accelerate the inhibition of thrombin, and abrogates HCII binding to thrombin exosite 1. While a minor portion of HCII 1-75 can be visualized in a crystallized HCII-thrombin S195A complex, the role of the rest of the extension is not well understood and the affinity of the HCII 1-75 interaction has not been quantitatively characterized. To address these issues, we expressed HCII 1-75 as a small, N-terminally hexahistidine-tagged polypeptide in E. coli. RESULTS: Immobilized purified HCII 1-75 bound active α-thrombin and active-site inhibited FPR-ck- or S195A-thrombin, but not exosite-1-disrupted γT-thrombin, in microtiter plate assays. Biotinylated HCII 1-75 immobilized on streptavidin chips bound α-thrombin and FPR-ck-thrombin with similar KD values of 330-340 nM. HCII 1-75 competed thrombin binding to chip-immobilized HCII 1-75 more effectively than HCII 54-75 but less effectively than the C-terminal dodecapeptide of hirudin (mean Ki values of 2.6, 8.5, and 0.29 µM, respectively). This superiority over HCII 54-75 was also demonstrated in plasma clotting assays and in competing the heparin-catalysed inhibition of thrombin by plasma-derived HCII; HCII 1-53 had no effect in either assay. Molecular modelling of HCII 1-75 correctly predicted those portions of the acidic extension that had been previously visualized in crystal structures, and suggested that an α-helix found between residues 26 and 36 stabilizes one found between residues 61-67. The latter region has been previously shown by deletion mutagenesis and crystallography to play a crucial role in the binding of HCII to thrombin exosite 1. CONCLUSIONS: Assuming that the KD value for HCII 1-75 of 330-340 nM faithfully predicts that of this region in intact HCII, and that 1-75 binding to exosite 1 is GAG-dependent, our results support a model in which thrombin first binds to GAGs, followed by HCII addition to the ternary complex and release of HCII 1-75 for exosite 1 binding and serpin mechanism inhibition. They further suggest that, in isolated or transferred form, the entire HCII 1-75 region is required to ensure maximal binding of thrombin exosite 1.

Murine ß-galactosidase stability is not dependent on temperature or protective protein/cathepsin A.

GM1 gangliosidosis, a neurodegenerative disorder, and Morquio B disease, a skeletal disorder, are lysosomal storage disorders caused by inherited defects in the enzyme ß-galactosidase (GLB1; EC 3.1.2.23; MIM #611458). Enzyme replacement therapy (ERT), a standard of care for a number of non-neuronopathic lysosomal storage disorders, is not yet available for GLB1 deficiency. Although functionally active recombinant human and feline GLB1 precursors have been purified, ERT has not yet been demonstrated in GM1 gangliosidosis or Morquio B disease models. A major obstacle to developing effective therapy may be the stability of human GLB1. We show here that mouse GLB1 has greater stability when compared to human GLB1, and that human GLB1 activity is temperature and protective-dependent on protein cathepsin A, while that of mouse GLB1 is not. These findings may impact on the eventual development of ERT for GLB1 deficiency. Despite our attempts to improve the extracellular stability of human GLB1 through sequence modification and the use of chemical chaperone N-butyldeoxygalactonojirimycin, the specific enzyme activity remained well below that of mGLB1.

Expression of cytokine genes following pre- and post-hatch immunization of chickens with herpesvirus of turkeys.

Induction of immune response as characterised by expression of cytokine genes in the spleen following immunization of pre- and post-hatch chickens with herpesvirus of turkeys (HVT) vaccine was studied. The pattern of expression of IFN-gamma and IL-10 genes in pre-hatch immunized chickens was different from that observed in post-hatch HVT immunized chickens. This expression pattern of cytokine genes was associated with significantly higher HVT transcripts in pre-hatch immunized chickens than in post-hatch immunized chickens. In conclusion, HVT immunization in chickens, irrespective of the age of immunization, stimulates host response characterised by the expression of cytokine genes, such as IFN-gamma and IL-10 in the spleen. However, the age of immunization appears to influence the temporal pattern of IFN-gamma and IL-10 expression as well as replication of HVT.

Molecular cloning and expression analysis of chicken MyD88 and TRIF genes.

Toll-like receptors (TLRs) trigger the innate immune system by responding to specific components of microorganisms. MyD88 and TRIF are Toll/interleukin (IL)-1 (TIR)-domain containing adapters, which play essential roles in TLR-mediated signalling via the MyD88-dependant and -independent pathways, respectively. Genes encoding several TLRs have been identified in the chicken genome, however, elements of their signalling pathways have not been well characterized. Here we describe the cloning of chicken MyD88 and TRIF orthologs, and examine the spatial and temporal expression of these genes. The chicken MyD88 cDNA was shown to have an open reading frame (ORF) of 1104 bp, encoding a predicted protein sequence of 368 aa, 8 aa short of a previously published coding sequence due to a premature stop codon. MyD88 gene expression was detected in each tissue tested except in muscle. The chicken TRIF cDNA possessed an ORF of 2205 bp, encoding a predicted protein sequence of 735 aa, which shared 37.3% similarity and 28.9% identity to human TRIF protein sequence. TRIF was ubiquitously expressed in all tissues.

Construction of a microarray specific to the chicken immune system: profiling gene expression in B cells after lipopolysaccharide stimulation.

The objective of this study was to profile gene expression in cells of the chicken immune system. A low-density immune-specific microarray was constructed that contained genes with known functions in the chicken immune system, in addition to chicken-expressed sequence tags (ESTs) homologous with mammalian immune system genes, which were systematically characterized by bioinformatic analyses. Genes and ESTs that met the annotation criteria were amplified and placed on a microarray. The microarray contained 84 immune system gene elements. As a means of calibration, the microarray was then used to examine gene expression in chicken B cells after lipopolysaccharide stimulation. Differential gene expression was observed at 6, 12, and 24 h but not at 48 h after stimulation. The results were validated by semiquantitative polymerase chain reaction. The microarray showed a high degree of reproducibility, as demonstrated by intra- and interassay correlation coefficients of 0.97 and 0.95, respectively. Thus, the low-density microarray developed in this study may be used as a tool for monitoring gene expression in the chicken immune system.