Solitary eosinophilic granulocytic sarcoma in a dog.

A 6-year-old male golden retriever presented with swelling of the left upper eyelid of 2 months duration, which did not improve following a course of antibiotics. Routine serum biochemistry, complete blood count and diagnostic imaging identified no clinically significant abnormalities. The mass was surgically excised, and histopathologic examination was performed. Eosinophilic granulocytic sarcoma (GS) was diagnosed based on the results of histopathology and immunohistochemistry. This is the first report of GS affecting the eyelid of a dog.

Mammalian Cell-Line-Expressed CD2v Protein of African Swine Fever Virus Provides Partial Protection against the HLJ/18 Strain in the Early Infection Stage.

A cell line expressing the CD2v protein of ASFV was generated. The efficient expression of CD2v protein was determined by immunofluorescence and Western blotting. The CD2v protein was Ni-affinity purified from the supernatant of cell cultures. The CD2v-expressing cells showed properties of hemadsorption, and the secreted CD2v protein exhibited hemagglutinating activity. The antigenicity and immunoprotection ability of CD2v were evaluated by immunizing pigs alone, combined with a cell-line-expressed p30 protein or triple combined with p30 and K205R protein. Immunized pigs were challenged with the highly virulent ASFV strain HLJ/18. Virus challenge results showed that CD2v immunization alone could provide partial protection at the early infection stage. Protein p30 did not show synergistic protection effects in immunization combined with CD2v. Interestingly, immunization with the triple combination of CD2V, p30 and K205R reversed the protection effect. The viremia onset time was delayed, and one pig out of three recovered after the challenge. The pig recovered from ASFV clinical symptoms, the rectal temperature returned to normal levels and the viremia was cleared. The mechanism of this protection effect warrants further investigation.

Paraganglioma of the Urinary Bladder in a Dog.

A 3-year-old male Bichon Frise developed lethargy, anorexia and haematuria. B-scan ultrasonography examination revealed a small, irregular, soft-textured mass in the bladder. Histopathologically, there was an incomplete fibrous pseudocapsule around the tumour tissue and although there was clear demarcation from the surrounding tissue, there was invasion of the capsule. Tumour cells proliferated in nests or cords of variable size, separated by fibrovascular tissue. The neoplastic cells were immunopositive for chromogranin A, synaptophysin and neuron-specific enolase, and electron microscopy revealed that they contained cytoplasmic secretory granules. On the basis of these findings, the tumour was diagnosed as a primary paraganglioma of the urinary bladder.

The IFN-γ-induced immunoproteasome is suppressed in highly pathogenic porcine reproductive and respiratory syndrome virus-infected alveolar macrophages.

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) evades cytotoxic T lymphocyte (CTL) responses through interactions between viral Nsp1α and Nsp4 and ß2 M heavy and light chains, respectively, of swine leukocyte antigen class (SLA)-I. However, whether the immunoproteasome (i-proteasome) complex, which is an important component of the antigen delivery pathway that functions by mediating peptide production, is also affected by viral infection is unknown. In this study, we investigated the effects of HP-PRRSV (HuN4-F5) infection on IFN-γ-induced i-proteasome expression using a cell culture system (alveolar macrophages, AMs). We found that this virus inhibited the expression of IFN-γ-induced i-proteasome subunits LMP2, LMP7, and MECL-1 at the mRNA and protein level. In addition, expression levels of the i-proteasome regulatory subunits PSME1 and PSME2 in the HP-PRRSV HuN4-F5-infected group were also significantly decreased compared to those in the uninfected group. However, there was no significant difference in the expression of proteasome subunits PSMB5, PSMB6, and PSMB7 between HP-PRRSV HuN4-F5-infected and uninfected groups. This study provides insight into the mechanisms underlying immune regulation by HP-PRRSV; specifically, this virus affects the antigen-processing machinery by suppressing IFN-γ-induced i-proteasome expression in infected AMs.

Induction of immunoproteasomes in porcine kidney (PK)-15 cells by interferon-γ and tumor necrosis factor-α.

Immunoproteasome (i-proteasome) has both immune and non-immune functions and plays important roles in controlling infections and combating illnesses. Our previous studies suggest that interferon (IFN)-γ induces the expression of three immune-specific catalytic subunits of the 20S proteasome that can replace their constitutive homologues to form the i-proteasome in immune cells, such as porcine alveolar macrophages (AMs) in vitro. However, i-proteasome levels and their modulation in non-immune cells such as the epithelial cells in pigs remain unknown. Here, we investigated the expression of i-proteasomes in non-immune cells (porcine kidney (PK)-15 cells) to determine i-proteasome modulation upon stimulation of PK-15 cells with IFN-γ and tumor necrosis factor (TNF)-α in vitro. The expression of i-proteasome subunits in PK-15 cells were regulated by IFN-γ and TNF-α. Remarkably, we found that the combination treatment of IFN-γ and TNF-α increased the expression of i-proteasome subunits LMP2, LMP7, and MECL-1 in PK-15 cells at transcriptional levels, but may decrease their expression at translational level, compared to their expression levels induced by individual cytokine treatments. These results provide critical insight into i-proteasome modulation in porcine non-immune cells, contribute further to our understanding of i-proteasome function in tissue pathogenesis and the development of antiviral adaptive immune responses against intracellular infections.

Transcriptome of porcine alveolar macrophages activated by interferon-gamma and lipopolysaccharide.

The molecular repertoire of porcine alveolar macrophages (PAMs) is greatly affected by the microenvironment they are exposed to, and specifically by inflammatory cytokines, such as interferon gamma (IFN-γ) released by activated lymphocytes, and microbial products, such as lipopolysaccharide (LPS). In our previous study, we found that IFN-γ- and LPS-activated PAMs (M1) could inhibit porcine reproductive and respiratory syndrome virus (PRRSV) replication. In this study, comprehensive analysis of the expression profiles of the genes associated with the polarization of M0-type PAMs (resting) toward M1 phenotypes (activated by IFN-γ and LPS) led to the following main results: 1) 1551 and 1823 genes were upregulated or downregulated in M1-type PAMs, respectively, compared with M0-type PAMs; 2) Among these, genes encoding ASS1 and CRTAM were the most upregulated and downregulated, respectively; 3) Genes involved in cytokine-cytokine receptor interaction and the JAK/STAT signaling pathway were significantly upregulated, suggesting their critical role in cellular activation; and 4) Genes involved in antigen proteolysis and presentation (immunoproteasome subunits), and inhibition of virus replication (host restriction factors) were significantly upregulated, emphasizing the critical role of these cytokines in immunity. Thus, our results provide important information for future studies on the role of PAM polarization in modulation of infection.

Anti-viral immune response in the lung and thymus: Molecular characterization and expression analysis of immunoproteasome subunits LMP2, LMP7 and MECL-1 in pigs.

Both the lung and the thymus are vital target organ for pathogens including viruses. The immunoproteasome (i-proteasome) enhances antigen presentation for MHC class I molecules to activate CD8+T lymphocyte. These facilitate antiviral adaptive immune response. Our previous study found that, expression of i-proteasome subunits in porcine lung was altered during normal and inflammatory conditions. To date, the expression of i-proteasome subunits in porcine thymus to viruses has not been investigated. In the present study, LMP2, LMP7, and MECL-1 were cloned, identified and their sequences encoded predicted proteins of 216, 275, and 278 amino acids, respectively. Expression of LMP2, LMP7, and MECL-1, in the cytoplasm and nucleus, was markedly altered in the porcine reproductive and respiratory syndrome virus (PRRSV)-infected lung and thymus. And dendritic cells and epithelial cells readily expressed the i-proteasome subunit LMP2 in the thymus of PRRSV-infected pigs compared to that in mock-infected pigs. Additionally, the in vitro stimulation of a PAM cell line with PolyI:C for 12 and 24 h resulted in increased LMP2, LMP7, and MECL-1 expression. These results suggest a central role for these complexes in the activation of an antiviral immune response in pigs. A better understanding of the role of the i-proteasome in different cell types, tissues, and hosts could improve vaccine design and facilitate the development of effective treatment strategies for viral infections.

Expression of immunoproteasome subunits in the porcine lung: Alterations during normal and inflammatory conditions.

The elimination of infected cells by cytotoxic T lymphocytes (CTLs) occurs through interactions between T cell receptors (TCRs) and pathogen-derived antigenic peptide-major histocompatibility complex (MHC) class I complexes. The immunoproteasome (i-proteasome), which is a large proteolytic machine derived from the constitutive proteasome, is highly efficient at processing antigens for presentation on MHC class I molecules to activate CD8+ T lymphocytes; this in turn facilitates antiviral adaptive immune responses. To date, i-proteasome expression in the porcine lung has not been investigated. In this study, we systematically analyzed the expression of the i-proteasome in vivo in the porcine lung and in vitro in alveolar macrophages (AMs) under normal and inflammatory conditions such as with IFN-γ stimulation or PRRSV infection. AMs were shown to readily express low levels of i-proteasome subunits, which were confined to the cytoplasm and nucleus under normal conditions. While i-proteasome expression could also be detected in other lung parenchymal cells including alveolar type I and II cells and bronchial epithelial cells during inflammatory conditions. Results showed that i-proteasome expression is markedly increased in IFN-γ-stimulated AMs and PRRSV-infected lung tissue. In addition, PRRSV infection promoted i-proteasome expression in AMs during the early stage of infection, and this was independent of IFN-γ; expression was attenuated during the later stage of infection in vitro. These results suggested that i-proteasome subunit expression can be induced in the porcine lung, which facilitates the development of antiviral adaptive immune responses against intracellular infections. These results could facilitate the development of therapeutics that target intracellular pathogens.

Sterile nodular panniculitis with lung and lymph node involvement in a Siberian tiger (Panthera tigris altica).

A 2- to 4-year-old uncastrated male Siberian tiger (Panthera tigris altica) bred in a local wild animal park presented with generalized clinical signs including abdominal pain, fever, lethargy, and anorexia, along with subcutaneous nodules along the trunk. The patient subsequently died of chronic, progressive dyspnea despite 45 days of antibiotic treatment. At necropsy, mesenteric fat inflammation and multiple subcutaneous, peritoneal, and intraabdominal nodules were observed. The lungs demonstrated congestion and heavy coagulation, and necrotic foci were observed on the cut surface. Histopathologically, the nodules were identified as granulomatous fatty tissue with numerous lymphocytes, infiltration with lipid-laden macrophages, and fibrosis. These changes were also noted in the lung. The etiology of this condition remains undetermined.

Efficacy evaluation of the C-strain-based vaccines against the subgenotype 2.1d classical swine fever virus emerging in China.

Classical swine fever (CSF) is a devastating infectious disease of pigs caused by classical swine fever virus (CSFV). The disease has been controlled following extensive vaccination with the lapinized attenuated vaccine C-strain for decades in China. However, frequent CSF outbreaks occurred recently in a large number of C-strain-vaccinated pig farms in China and a new subgenotype 2.1d of CSFV has been reported to be responsible for the outbreaks. Here we analyzed the molecular variations and antigenic differences among the C-strain-based commercial vaccines of different origins from different manufacturers in China, and reevaluated the vaccines against the emerging subgenotype 2.1d strain of CSFV. The results showed that the C-strain adapted to the continuous ST cell line (CST) contain a unique M290K variation on the E2 protein, compared to those of primary BT cells (CBT) or rabbit origin (CRT) and the traditional C-strain sequences available in the GenBank database. Serum neutralization test revealed the antigenic differences between CST and CBT or CRT. Notably, the neutralizing titers of porcine anti-C-strain sera against the CSFV isolate of subgenotype 2.1d were significantly lower than those against C-strain or Shimen strain. The C-strain-vaccinated, subgenotype 2.1d HLJZZ2014 strain-challenged pigs did not show any clinical signs and all survived. However, these pigs displayed mild pathological and histological lesions, and the CSFV viral RNA was detected in the various tissue and blood samples. Taken together, the C-strain-based vaccines of different origins showed molecular variations and antigenic differences, and could provide clinical but not pathological and virological protection against the subgenotype 2.1d CSFV emerging in China. Further investigation is needed to comprehensively assess the efficacy of C-strain of different doses against the subgenotype 2.1d CSFV.

Infection with equine infectious anemia virus vaccine strain EIAVDLV121 causes no visible histopathological lesions in target organs in association with restricted viral replication and unique cytokine response.

The live equine infectious anemia virus (EIAV) vaccine strain EIAVDLV121 was developed by in vitro attenuation of a virulent strain, EIAVLN40, in the 1970s, and it has been demonstrated to induce protective immunity under laboratory and natural EIAV infection conditions. The detailed biological features of this attenuated virus remain to be further investigated. Experimental inoculation with EIAVDLV121 did not result in clinical symptoms even with immunosuppressive treatment in our previous studies. Here, we further investigated whether the replication of the vaccine strain EIAVDLV121 in experimentally infected horses causes histopathological lesions to develop in the targeted organs. Both the lungs and the spleen have been demonstrated to support EIAV replication. By evaluating the gross macroscopic and histological changes, we found that EIAVDLV121 did not cause detectable histopathological lesions and that it replicated several hundred times more slowly than its parental virulent strain, EIAVLN40, in tissues. Immunochemical assays of these tissues indicated that the primary target cells of EIAVDLV121 were monocytes/macrophages, but that EIAVLN40 also infected alveolar epithelial cells and vascular endothelial cells. In addition, both of these viral strains promoted the up- and down-regulation of the expression of various cytokines and chemokines, implicating the potential involvement of these cellular factors in the pathological outcomes of EIAV infection and host immune responses. Taken together, these results demonstrate that the EIAV vaccine strain does not cause obvious histopathological lesions or clinical symptoms and that it induces a unique cytokine response profile. These features are considered essential for EIAVDLV121 to function as an effective live vaccine.

Dietary curcumin supplementation attenuates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in C57BL mice.

Studies in vivo and in vitro suggest that curcumin is a neuroprotective agent. Experiments were conducted to determine whether dietary supplementation with curcumin has neuroprotective effects in a mouse model of Parkinson's disease (PD). Treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) significantly induced the loss of dopaminergic cells in the substantia nigra and deletion of dopamine in the striatum, which was attenuated by long-term (7 weeks) dietary supplementation with curcumin at a concentration of 0.5% or 2.0% (w/w). Although curcumin did not prevent the MPTP-induced apoptosis of neuroblasts in the subventricular zone (SVZ), it promoted the regeneration of neuroblasts in the anterior part of the SVZ (SVZa) at 3 days after MPTP treatment. Furthermore, curcumin enhanced the MPTP-induced activation of microglia and astrocytes in the striatum and increased the expression of glial cell line-derived neurotrophic factor (GDNF) and transforming growth factor-ß1 (TGFß1) in the striatum and SVZ. GDNF and TGFß1 are thought to play an important role in protecting neurons from injury in the central and peripheral nervous systems. These results suggest that long-term administration of curcumin blocks the neurotoxicity of MPTP in the nigrostriatal dopaminergic system of the mouse and that the neuroprotective effect might be correlated with the increased expression of GDNF and TGFß1. Curcumin may be effective in preventing or slowing the progression of PD.

Transiently impaired neurogenesis in MPTP mouse model of Parkinson's disease.

It is still being debated whether neurogenesis in the subventricular zone (SVZ) is enhanced in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injury in the adult mouse brain. Our previous studies provided evidence that MPTP induces apoptosis of migrating neuroblasts (neural progenitor cells, A cells) in the SVZ and rostral migratory stream (RMS). We investigated cellular kinetics in the adult SVZ and olfactory bulb (OB) after MPTP damage. Cells were labeled with bromodeoxyuridine (BrdU), and the effects of MPTP on the survival and fate of migrating and residing neuroblasts were evaluated. Two days after BrdU labeling and MPTP treatment, the number of BrdU-positive cells in the SVZ and OB of MPTP-treated mice was significantly lower than in the SVZ and OB of saline controls. Additionally, fewer BrdU-positive cells migrated to the OB of treated mice than to that of saline controls, and the cells that did migrate diffused radially into the granule cell layer (GCL) when observed at 7, 14, and 28 days. In the OB GCL, the differentiation of BrdU-positive cells into mature neurons significantly attenuated 14 and 28 days after MPTP injury. Moreover, the impaired neurogenesis was followed by a recovery of A cells in the SVZ and OB, suggesting activation of the self-repair process as a result of MPTP-induced depletion of BrdU-positive cells. Our findings clarify the kinetics underlying neurogenesis in MPTP-treated mice and may contribute to the development of an animal model of Parkinson's disease, and the demonstration of cellular kinetics in SVZ may also provide a new insight into assessing neurogenesis in MPTP-treated mouse.

Characterization of Equine Infectious Anemia Virus Integration in the Horse Genome.

Human immunodeficiency virus (HIV)-1 has a unique integration profile in the human genome relative to murine and avian retroviruses. Equine infectious anemia virus (EIAV) is another well-studied lentivirus that can also be used as a promising retro-transfection vector, but its integration into its native host has not been characterized. In this study, we mapped 477 integration sites of the EIAV strain EIAVFDDV13 in fetal equine dermal (FED) cells during in vitro infection. Published integration sites of EIAV and HIV-1 in the human genome were also analyzed as references. Our results demonstrated that EIAVFDDV13 tended to integrate into genes and AT-rich regions, and it avoided integrating into transcription start sites (TSS), which is consistent with EIAV and HIV-1 integration in the human genome. Notably, the integration of EIAVFDDV13 favored long interspersed elements (LINEs) and DNA transposons in the horse genome, whereas the integration of HIV-1 favored short interspersed elements (SINEs) in the human genome. The chromosomal environment near LINEs or DNA transposons potentially influences viral transcription and may be related to the unique EIAV latency states in equids. The data on EIAV integration in its natural host will facilitate studies on lentiviral infection and lentivirus-based therapeutic vectors.

Mice transgenic for equine cyclin T1 and ELR1 are susceptible to equine infectious anemia virus infection.

BACKGROUND: As a member of the tumor necrosis factor receptor (TNFR) protein superfamily, equine lentivirus receptor 1 (ELR1) has been shown to be expressed in various equine cells that are permissive for equine infectious anemia virus (EIAV) replication. The EIAV Tat protein (eTat) activates transcription initiated at the viral long terminal repeat (LTR) promoter through a unique mechanism that requires the recruitment of the equine cyclin T1 (eCT1) cofactor into the viral TAR RNA target element. In vitro studies have demonstrated that mouse fibroblast cell lines (e.g., NIH 3T3 cells) that express the EIAV receptor ELR1 and eCT1 support the productive replication of EIAV. Therefore, we constructed transgenic eCT1- and ELR1-expressing mice to examine whether they support in vivo EIAV replication. FINDINGS: For the first time, we constructed mice transgenic for ELR1 and eCT1. Real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis confirmed that ELR1 and eCT1 were expressed in the transgenic mouse tissues, particularly in the intestines, spleen and lymph nodes. Consistent with the results of EIAV infection in NIH 3T3 cells expressing ELR1 and eCT1, mouse embryonic fibroblasts (MEFs) from the transgenic mice could support EIAV replication. More importantly, this virus could infect and replicate in mouse blood monocyte-derived macrophages (mMDMs). Macrophages are the principle target cell of EIAV in its natural hosts. Furthermore, after the transgenic mice were inoculated with EIAV, the virus could be detected not only in the plasma of the circulating blood but also in multiple organs, among which, the spleen and lymph nodes were the predominant sites of EIAV replication. Finally, we found that consistent with high viral replication levels, the relevant pathological changes occurred in the spleen and lymph nodes. CONCLUSIONS: Our results show that mice transgenic for ELR1 and eCT1 are susceptible to EIAV infection and replication. Further, EIAV infection can cause lesions on the spleen and lymph nodes, similar to those frequently observed in horses, the natural hosts. Therefore, ELR1 and eCT1 are essential in vivo for EIAV invasion and replication.

Gene expression profiles in the fetal mouse brain after etoposide (VP-16) administration.

The aim of this study was to analyze the response of gene expression caused by etoposide (VP-16) in the fetal mouse brain. Four miligrams/kilogram of VP-16 was intraperitoneally injected into pregnant mice on day 12 of gestation (GD 12). Gene expression profiling of the VP-16-treated fetal mouse brain by DNA microarray was performed. The expression changes of the target genes of p53 were also examined by real-time RT-PCR. VP-16 induced S-phase accumulation, G2/M arrest, and eventually apoptosis of neuroepithelial cells in the fetal brain. DNA microarray analysis revealed that 8 of cell cycle control- and apoptosis-related genes were upregulated and that 5 of DNA damage, repair, replication, and transcription genes were also upregulated in the fetal telencephalons at 4 h after VP-16 treatment (HAT). The results of real-time RT-PCR demonstrated that the expression of topoisomerase IIα was increased at 4 and 8 HAT. The expression of pro-apoptotic factors such as puma, noxa, bax, and cyclin G was also increased from 4 to 12 HAT. These results suggest that VP-16 induces DNA damage, DNA repair, cell cycle alternation, and apoptosis in the fetal mouse brain. In addition, VP-16-induced apoptosis is mediated through the mitochondrial pathway in a p53-related manner. The present study will provide a better understanding of the mechanisms of VP-16-induced fetal brain injury.

Resistance of the golden hamster to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-neurotoxicity is not only related with low levels of cerebral monoamine oxidase-B.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been proved to be a potent neurotoxin on dopaminergic neurons inducing most of the symptoms and cerebral lesions observed in the idiopathic Parkinson's disease (PD). Although there is a substantial body of theory and researches about the effects of MPTP on susceptible mice and nonhuman primates, there are only few studies in resistant animals, such as golden hamsters (GH). The low levels of cerebral monoamine oxidase-B (MAO-B) enzyme have been proposed as the cause of the GH insensitivity to MPTP. The aim of this study was to elucidate whether MAO-B is the only factor which confer GH resistance to MPTP. Neither loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) nor cell death in the subventricular zone (SVZ) were found in female GH in response to an acute intraperitoneal (ip) MPTP treatment. To prove the role of MAO-B in the MPTP-resistance, female and male GH was intracerebroventricularly (icv) injected with either MPTP or 1-methyl-4-phenylpyridinum (MPP(+)). Neither depletion in the number of dopaminergic neurons, nor astrogliosis, cell death in the SVZ of female and male GH were registered after an icv treatment with MPTP or MPP(+). Furthermore, we demonstrated that MAO-B is located predominantly within the endothelial cells in the blood brain barrier (BBB), but not in the astroglia. The present results raise the possibility that, in GH, other mechanisms, apart from the low levels of regional MAO-B, confer resistance to MPTP and its metabolites.

Changes in blood parameters and the expression of coagulation-related genes in lactating Sprague-Dawley rats.

This study measured blood parameters, particularly those related to coagulation, and alterations in the expression levels of blood-coagulation-related genes in lactating Sprague-Dawley rats. The day of delivery was designated as lactation day 0 (LD 0). On the day after delivery (LD 1), prothrombin time and overall activity of vitamin-K-dependent coagulation factors were decreased, whereas fibrinogen contents, platelet counts and antithrombin III concentrations were increased as compared with those in nonpregnant rats. In addition, hepatic expression of blood-coagulation-related genes in the liver was increased at LD 0 as compared with that in nonpregnant rats. These changes may be physiologic responses to prevent prolonged bleeding at delivery. Except for fibrinogen content, which remained elevated, the described changes returned to baseline on and after LD 7. Activities of AST, ALT, and ALP were increased on LD 7, 14, and 21 as compared with nonpregnant rats. In contrast, total protein, albumin, Cl, and Ca were consistently lower on LD 7, 14, or 21 as compared with levels in nonpregnant rats. These results provide background data for evaluation of nursing rats.

MPTP-induced neuroblast apoptosis in the subventricular zone is not regulated by dopamine or other monoamine transporters.

For 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to exert neurotoxicity on dopaminergic neurons, 1-methyl-4-phenylpyridinium (MPP+), a metabolite of MPTP, must be taken up into the dopaminergic neuron via the dopamine transporter (DAT). Previous reports have shown that MPTP also causes neuroblast apoptosis in the subventricular zone (SVZ) of adult mice. The aim of this study is to elucidate the role of DAT and other monoamine transporters including vesicular monoamine transporter 2 (VMAT2), the serotonin transporter (SERT), and the norepinephrine transporter (NET) on the neuroblast apoptosis induced by MPTP administration. There were no DAT-positive neuroblasts in the SVZ, whereas some neuroblasts were immunopositive for VMAT2 and SERT. To examine whether these transporters are involved in MPTP-induced neuroblast apoptosis in the SVZ, terminal deoxynucleotidyl transferase-mediated dUTP endlabeling (TUNEL)-positive cells were semiquantitatively analyzed after the injection of GBR12909 (GBR), a DAT inhibitor; tetrabenazine (TBZ), a VMAT2 inhibitor; fluoxetine (FLU), a SERT inhibitor, or desipramine (DES), a NET inhibitor, prior to MPTP injection. However, the injection of these transporter inhibitors had no influence on the MPTP-induced neuroblast apoptosis in the SVZ. It is likely that neither DAT nor other monoamine transporters are involved in MPTP-induced neuroblast apoptosis. The present findings suggest that the neurotoxicity of MPTP to neuroblasts in the SVZ does not require DAT or other monoamine transporters, and the apoptosis it induces may be executed through other unknown pathways.

Changes in blood parameters and coagulation-related gene expression in pregnant rats.

The present study examined changes in maternal blood parameters, particularly those related to blood coagulation, as well as alterations in blood coagulation-related gene expression in the liver during gestation in rats. Fibrinogen concentration and platelet count increased as pregnancy progressed whereas prothrombin time and overall activity of vitamin-K-dependent coagulation factors decreased before delivery, suggesting a physiologic response to prevent prolonged bleeding at parturition. Conversely, compared with values for nonpregnant rats, activated partial thromboplastin time was prolonged before delivery and antithrombin time was significantly higher during fetal organogenesis and thereafter, indicating a mechanism to prevent the development of deep tissue thrombosis in dams. DNA microarray analysis revealed no differences in coagulation-related gene expression in the liver on gestation day 13 between pregnant and nonpregnant rats, whereas the gene expression of various fibrinogen-related factors, coagulation factors II and X, and the anticoagulation factor-related factor leuserpin 2 were increased on gestational day 19. In addition, changes similar to those reported previously in pregnant rats were confirmed. The data obtained from the present study can be used as background data for effective evaluation of reproductive toxicology in rats, and they suggest that the rat is a useful animal model for investigating the mechanisms of disorders in the blood coagulation system that can occur during late pregnancy in women.