Detecting population structure in a high gene-flow species, Atlantic herring (Clupea harengus): direct, simultaneous evaluation of neutral vs putatively selected loci.

In many marine fish species, genetic population structure is typically weak because populations are large, evolutionarily young and have a high potential for gene flow. We tested whether genetic markers influenced by natural selection are more efficient than the presumed neutral genetic markers to detect population structure in Atlantic herring (Clupea harengus), a migratory pelagic species with large effective population sizes. We compared the spatial and temporal patterns of divergence and statistical power of three traditional genetic marker types, microsatellites, allozymes and mitochondrial DNA, with one microsatellite locus, Cpa112, previously shown to be influenced by divergent selection associated with salinity, and one locus located in the major histocompatibility complex class IIA (MHC-IIA) gene, using the same individuals across analyses. Samples were collected in 2002 and 2003 at two locations in the North Sea, one location in the Skagerrak and one location in the low-saline Baltic Sea. Levels of divergence for putatively neutral markers were generally low, with the exception of single outlier locus/sample combinations; microsatellites were the most statistically powerful markers under neutral expectations. We found no evidence of selection acting on the MHC locus. Cpa112, however, was highly divergent in the Baltic samples. Simulations addressing the statistical power for detecting population divergence showed that when using Cpa112 alone, compared with using eight presumed neutral microsatellite loci, sample sizes could be reduced by up to a tenth while still retaining high statistical power. Our results show that the loci influenced by selection can serve as powerful markers for detecting population structure in high gene-flow marine fish species.

Temporally stable genetic structure of heavily exploited Atlantic herring (Clupea harengus) in Swedish waters.

Information on the temporal stability of genetic structures is important to permit detection of changes that can constitute threats to biological resources. Large-scale harvesting operations are known to potentially alter the composition and reduce the variability of populations, and Atlantic herring (Clupea harengus) has a long history of heavy exploitation. In the Baltic Sea and Skagerrak waters, the census population sizes have declined by 35-50% over the last three decades. We compared the genetic structure of Atlantic herring in these waters sampled at least two different times between 1979 and 2003 by assaying 11 allozyme and nine microsatellite loci. We cannot detect any changes in the amount of genetic variation or spatial structure, and differentiation is weak with overall F(ST)=0.003 among localities for the older samples and F(ST)=0.002 for the newer ones. There are indications of temporal allele frequency changes, particularly in one of five sampling localities that is reflected in a relatively small local N(e) estimate of c. 400. The previously identified influence of selection at the microsatellite locus Cpa112 remains stable over the 24-year period studied here. Despite little genetic differentiation, migration among localities appears small enough to permit demographic independence between populations.

Porcine neural xenografts in rats and mice: donor tissue development and characteristics of rejection.

Embryonic ventral mesencephalic tissue from the pig is a potential alternative donor tissue for neural transplantation to Parkinson's disease patients. For stable graft survival, the host immune response has to be prevented. This study was performed in order to analyze the mechanisms and dynamics of neural xenograft rejection, as well as neurobiological properties of the donor tissue. Adult normal mice and rats, and cyclosporin A-treated rats, received intrastriatal transplants of dissociated embryonic ventral mesencephalic pig tissue that was 27 or 29 embryonic days of age (E27 and E29). The animals were perfused at 2, 4, 6, and 12 weeks after grafting and the brains were processed for immunohistochemistry of dopaminergic (tyrosine hydroxylase positive) neurons, CD4(+) and CD8(+) lymphocytes, natural killer cells, macrophages, microglia, and astrocytes. Thirty-five rats received daily injections of BrdU for 5 consecutive days at different time points after transplantation and were perfused at 6 weeks. These animals were analyzed for proliferation of cells in the donor tissue, both in healthy and in rejecting grafts. No tyrosine hydroxylase-positive cells proliferated after grafting. Our results demonstrated that E27 was superior to E29 donor tissue for neurobiological reasons. Cyclosporin A immunosuppression was protective only during the first weeks and failed to protect the grafts in a long-term perspective. Grafts in mice were invariably rejected between 2 and 4 weeks after transplantation, while occasional grafts in untreated rats survived up to 12 weeks without signs of an ongoing rejection process. CD8(+) lymphocytes and microglia cells are most likely important effector cells in the late, cyclosporin A-resistant rejection process.

Enhanced survival of porcine neural xenografts in mice lacking CD1d1, but no effect of NK1.1 depletion.

Transplantation of embryonic porcine neurons may restore neurological function in patients with Parkinson's disease, if immunological rejection could be prevented. This study was performed to investigate the role of natural killer cells (NK cells) and NK1.1+ T cells (NK T cells) in the rejection of neural xenografts. A cell suspension was prepared from the ventral mesencephalon of 26-27-day-old pig embryos, and 2 microl was implanted in the right striata of mutant CD1d1 null (CD1.1-/-) mice, NK1.1-depleted mice, and controls. The CD1.1-/- mice are deficient in NK T cells and the antigen-presenting molecule CD1d1. Graft survival and host responses were determined immunohistochemically using markers for dopamine neurons, CD4-, CD8- cells, microglia, and macrophages. At 2 weeks, the grafts were significantly larger in CD1.1-/- mice, 0.09 +/- 0.02 microl (mean +/- SEM), compared with controls, 0.05 +/- 0.01 microl. There was no significant difference between NK1.1-depleted mice, 0.02 +/- 0.01 microl, and controls. At 5 weeks, two grafts were still present in the CD1-/- mice, whereas only scars remained in the controls and in the NK1.1-depleted mice. Immune reactions were strong at 2 weeks and less pronounced at 5 weeks in all groups. Microglial activation was lower in NK-depleted mice than in the controls at 2 weeks. In contrast to organ xenografting, NK1.1+ cells do not seem to be important mediators of the rejection of discordant cellular neural xenografts. However, our results suggest that the antigen-presenting molecule CD1d1 may be involved in the rejection process.

Effects of immunosuppressive treatment on host responses against intracerebral porcine neural tissue xenografts in rats.

BACKGROUND: Embryonic xenogeneic neural tissue is an alternative for transplantation in Parkinson's disease, but immune responses limit the application. The aims of this study were to enhance the in vitro viability rates by donor tissue pretreatment; to compare the efficacy of cyclosporine A (CsA) and tacrolimus (FK) in inhibiting xenograft rejection in rats; to evaluate additional inductive therapy with prednisolone (PRE) or mycophenolate mofetil (MMF). METHODS: Tirilazad (a lipid peroxidase inhibitor) or FK and acYVAD-cmk (a caspase inhibitor), were added to embryonic porcine ventral mesencephalic tissue and viability was assessed in vitro. Tirilazad-treated tissue was grafted to the striatum of rats that were either left untreated or immunosuppressed with FK (1 mg/kg) or CsA (15 mg/kg) alone or in combination with a 2-week PRE (20 mg/kg) or MMF (40 mg/kg) induction course. Xenograft survival and host responses were determined using immunohistochemistry. RESULTS: Pretreatment with tirilazad enhanced tissue survival in vitro. After transplantation into untreated controls, there was no graft survival at twelve weeks. Neural cell counts were significantly improved in immunosuppressed recipients, but there were no differences between the treatment groups. Additional inductive treatment reduced the infiltration with CD4+ and CD8+ cells, and macrophage infiltration was reduced compared with animals given CsA or FK alone. CONCLUSION: Pretreatment of the donor tissue with free-radical scavengers reduces cell loss caused by tissue trauma. Porcine neural tissue xenografts survive significantly better in animals immunosuppressed with either FK or CsA. Additional inductive treatment with PRE or MMF reduced the infiltration of host cells into the xenografts.

Neural tissue xenografting.

Neural transplantation may become an important treatment alternative for focal brain disorders. To date, the most successful grafts have been obtained in patients with Parkinson's disease. Completely normalized dopamine production and reduction of Parkinsonian symptoms have been demonstrated 10 years after grafting. However, the allogeneic donor tissue has to be obtained from induced abortions, and there are logistical difficulties, risks of infection, and ethical constraints limiting a wider clinical use. Xenografting is an alternative that could bridge these limitations if immunological rejection could be prevented. Pig embryonic neural tissue has been grafted to patients with Parkinson's disease, but no functional benefits have clinically been proven so far. The immune reactions to neural xenografts were incompletely characterized at the time of these early clinical trials, and it is likely that the treatments used were insufficient and that the grafts were rejected. In this article we will review new experiments addressing the immune responses against porcine neural tissue grafted to the adult brain, including the role of antibodies, complement, natural killer (NK) cells, lymphocytes, as well as the effects of immunosuppressive drugs and donor tissue modifications.

Intrastriatal ventral mesencephalic xenografts of porcine tissue in rats: immune responses and functional effects.

Transplantation of neural tissue from other species has the potential to improve function in patients with neurodegenerative disorders. We investigated the functional effects of embryonic porcine dopaminergic neurons transplanted in a rat model of Parkinson's disease and the immune responses to the grafts in immunosuppressed and nonimmunosuppressed hosts. Twenty-three rats with unilateral 6-hydroxydopamine lesions received dissociated, 27-day-old embryonic porcine ventral mesencephalic tissue in the right striatum. Eighteen rats received cyclosporine (10 mg/kg, IP, daily) during the whole period of 14 weeks, in combination with prednisolone (20 mg/kg, IP, daily) the first 4 days. Five rats served as nonimmunosuppressed controls. All rats were tested for amphetamine-induced rotational behavior at 3-week intervals. Two immunosuppressed rats were excluded due to severe side effects of the treatment. Functional recovery was seen in 9 of 16 immunosuppressed rats at 12 weeks. Six animals remained functionally recovered at 14 weeks and contained an average of 5750+/-1450 (SEM) dopaminergic neurons. Between 9 and 14 weeks, three immunosuppressed rats rejected their grafts, based on rotation scores and immunohistochemical demonstration of cell infiltrates. One additional immunosuppressed rat showed evidence of ongoing rejection at 14 weeks. The striata in animals with ongoing or recent rejection contained large numbers of CD4- and CD8-positive lymphocytes, NK cells, macrophages, and microglia cells, whereas scar tissue was found in rats with grafts rejected at earlier time points (n = 11). Embryonic porcine ventral mesencephalic tissue matures in the adult rat striatum, reinnervates the host brain, and restores behavioral defects. Immunosuppressive treatment was necessary for long-term graft survival and functional recovery, but did not sufficiently protect from rejection mechanisms. Porcine neural tissue is an interesting alternative to embryonic human tissue for intracerebral transplantation in neurodegenerative diseases. However, to achieve stable graft survival in discordant xenogeneic combinations, an appropriate immunosuppressive treatment or donor tissue modifications are needed.

Discordant neural tissue xenografts survive longer in immunoglobulin deficient mice.

BACKGROUND: The immune response against discordant xenografts in the brain is incompletely understood and remains a major obstacle for future clinical applications of xenogeneic neural tissue transplants in neurodegenerative disorders. To determine the role of antibodies in the rejection process, we compared graft survival and immune reactions between immunoglobulin deficient (IgKO) and normal mice. METHODS: A cell suspension of embryonic porcine ventral mesencephalon was injected into the striatum of adult normal and IgKO mice. Graft sizes and number of infiltrating CD4- and CD8-positive lymphocytes were determined by stereological methods at 4 days and 2, 4, and 6 weeks after the transplants. Microglial accumulation was determined using the optical densitometrical method. Intraparenchymal deposition of IgG was investigated at 4 days and 2 weeks. RESULTS: The majority of IgKO mice had surviving grafts for up to 4 weeks, whereas survival was minimal in control mice beyond 4 days. Graft sizes differed significantly between IgKO and control mice at 2 weeks (P<0.01, Kruskal Wallis ANOVA, followed by Mann Whitney test). The majority of infiltrating lymphocytes were CD4-positive in control mice but CD8-positive in IgKO mice. Microglial accumulation was strong around surviving grafts in IgKO mice at 4 weeks. Prominent staining of IgG, diffuse in the transplanted hemisphere and specific on grafted neurons, was found in control mice. CONCLUSIONS: Our results suggest that immunoglobulins play an initiating role in rejection of discordant neural xenografts. After a prolonged graft survival of approximately 4 weeks, a cellular response with a large proportion CD8-positive cells leads to rejection in IgKO mice.

Discordant xenografts: different outcome after mouse and rat neural tissue transplantation to guinea-pigs.

Embryonic neural tissue obtained from other species has been considered as a donor tissue source in repair strategies for human neurodegenerative disorders. The neuro- and immunobiology of distantly related species combinations, discordant xenografts, need to be characterised. For this purpose, a small animal model would be an important research tool. Adult guinea-pigs, and adult rats as controls, received intrastriatal grafts of either mouse or rat embryonic ventral mesencephalic tissue. The survival rates and types of host immune response were assessed at 2 weeks after grafting using stereological techniques and semi-quantitative evaluations. In the mouse-to-guinea-pig group, all transplants were rejected and no tyrosine hydroxylase-immuno reactive (TH-IR) cells remained. In the rat-to-guinea-pig group, there was good survival of TH-IR cells (5050 SEM+/-1550), similar to that in the rat-to-rat group (4900 SEM+/-1540). In the mouse-to-rat group, half of the animals had no surviving TH-IR cells (520 SEM+/-230 for the whole group). These species combinations offer inexpensive, efficient, and suitable conditions to study important survival factors for discordant xenogeneic neural tissue transplants. The factors responsible for the divergent graft outcomes between the two combinations might provide clues on how to manipulate xenogeneic tissue to increase survival rates in the future.