Unique structure of ozoralizumab, a trivalent anti-TNFα NANOBODY® compound, offers the potential advantage of mitigating the risk of immune complex-induced inflammation.

Biologics have become an important component of treatment strategies for a variety of diseases, but the immunogenicity of large immune complexes (ICs) and aggregates of biologics may increase risk of adverse events is a concern for biologics and it remains unclear whether large ICs consisting of intrinsic antigen and therapeutic antibodies are actually involved in acute local inflammation such as injection site reaction (ISR). Ozoralizumab is a trivalent, bispecific NANOBODY® compound that differs structurally from IgGs. Treatment with ozoralizumab has been shown to provide beneficial effects in the treatment of rheumatoid arthritis (RA) comparable to those obtained with other TNFα inhibitors. Very few ISRs (2%) have been reported after ozoralizumab administration, and the drug has been shown to have acceptable safety and tolerability. In this study, in order to elucidate the mechanism underlying the reduced incidence of ISRs associated with ozoralizumab administration, we investigated the stoichiometry of two TNFα inhibitors (ozoralizumab and adalimumab, an anti-TNFα IgG) ICs and the induction by these drugs of Fcγ receptor (FcγR)-mediated immune responses on neutrophils. Ozoralizumab-TNFα ICs are smaller than adalimumab-TNFα ICs and lack an Fc portion, thus mitigating FcγR-mediated immune responses on neutrophils. We also developed a model of anti-TNFα antibody-TNFα IC-induced subcutaneous inflammation and found that ozoralizumab-TNFα ICs do not induce any significant inflammation at injection sites. The results of our studies suggest that ozoralizumab is a promising candidate for the treatment of RA that entails a lower risk of the IC-mediated immune cell activation that leads to unwanted immune responses.

Repeated administration of acrylamide for 28 days suppresses adult neurogenesis of the olfactory bulb in young-adult rats.

Acrylamide (AA) is a neurotoxicant that inhibits synaptic function in distal axons. We previously found that AA decreased neural cell lineages during late-stage differentiation of adult hippocampal neurogenesis and downregulated genes related to neurotrophic factor, neuronal migration, neurite outgrowth, and synapse formation in the hippocampal dentate gyrus in rats. To investigate whether olfactory bulb (OB)-subventricular zone (SVZ) neurogenesis is similarly affected by AA exposure, AA was administered to 7-week-old male rats via oral gavage at doses of 0, 5, 10, and 20 mg/kg for 28 days. Immunohistochemical analysis revealed that AA decreased the numbers of doublecortin-positive (+) cells and polysialic acid-neural cell adhesion molecule+ cells in the OB. On the other hand, the numbers of doublecortin+ cells and polysialic acid-neural cell adhesion molecule+ cells in the SVZ did not change with AA exposure, suggesting that AA impaired neuroblasts migrating in the rostral migratory stream and OB. Gene expression analysis in the OB revealed that AA downregulated Bdnf and Ncam2, which are related to neuronal differentiation and migration. These results suggest that AA decreased neuroblasts in the OB by suppressing neuronal migration. Thus, AA decreased neuronal cell lineages during late-stage differentiation of adult neurogenesis in the OB-SVZ, similar to the effect on adult hippocampal neurogenesis.

Repeated administration of acrylamide for 28 days reduces late-stage progenitor cells and immature granule cells accompanying impaired neurite outgrowth in the adult hippocampal neurogenesis in young-adult rats.

Acrylamide (AA) is a neurotoxicant that causes synaptic impairment in distal axons. We previously found that developmental exposure to AA decreased proliferation of late-stage neural progenitor cells (NPCs) in the hippocampal neurogenesis of the dentate gyrus (DG) in rats. To investigate whether hippocampal neurogenesis is similarly affected by AA exposure in a general toxicity study, AA was administered to 7-week-old male rats via oral gavage at dosages of 0, 5, 10, and 20 mg/kg for 28 days. In the subgranular zone (SGZ) and granule cell layer, AA decreased the densities of doublecortin-positive (+) cells and TOAD-64/Ulip/CRMP protein 4b+ cells per SGZ length. In addition, AA decreased the neurite length of doublecortin+ cells and downregulated genes related to neurite outgrowth (Ncam2 and Nrep) and neurotrophic factor (Bdnf and Ntrk2) in the DG. These results suggest that AA exposure for 28 days decreases type-3 NPCs and immature granule cells in neurogenesis of granule cell lineages involving the impairment of neurite outgrowth in young-adult rats. In the DG hilus, AA increased the density of cholinergic receptor nicotinic beta 2 subunit+ cells. AA also downregulated Reln related to the control of neuronal migration by interneurons in the DG. Furthermore, AA decreased the density of glial fibrillary acidic protein (GFAP)+ astrocytes in the DG hilus and downregulated Gfap and the genes of oligodendrocyte progenitor cells (Cspg4 and Pdgfra). Thus, AA decreased granule cell lineage subpopulations in the late-stage differentiation of hippocampal neurogenesis after young-adult stage exposure, exhibiting a pattern similar to the developmental exposure.

A novel anti-TNF-α drug ozoralizumab rapidly distributes to inflamed joint tissues in a mouse model of collagen induced arthritis.

In clinical studies, the next-generation anti-tumor necrosis factor-alpha (TNF-α) single domain antibody ozoralizumab showed high clinical efficacy shortly after the subcutaneous injection. To elucidate the mechanism underlying the rapid onset of the effects of ozoralizumab, we compared the biodistribution kinetics of ozoralizumab and adalimumab after subcutaneous injection in an animal model of arthritis. Alexa Fluor 680-labeled ozoralizumab and adalimumab were administered by subcutaneous injection once (2 mg/kg) at five weeks after induction of collagen-induced arthritis (CIA) in an animal arthritis model. The time-course of changes in the fluorescence intensities of the two compounds in the paws and serum were evaluated. The paws of the CIA mice were harvested at four and eight hours after the injection for fluorescence microscopy. Biofluorescence imaging revealed better distribution of ozoralizumab to the joint tissues than of adalimumab, as early as at four hours after the injection. Fluorescence microscopy revealed a greater fluorescence intensity of ozoralizumab in the joint tissues than that of adalimumab at eight hours after the injection. Ozoralizumab showed a significantly higher absorption rate constant as compared with adalimumab. These results indicate that ozoralizumab enters the systemic circulation more rapidly and is distributed to the target tissues earlier and at higher levels than conventional IgG antibodies. Our investigation provides new insight into the mechanism underlying the rapid onset of the effects of ozoralizumab in clinical practice.

Oral exposure to high-dose ethanol for 28 days in rats reduces neural stem cells and immediate nascent neural progenitor cells as well as FOS-expressing newborn granule cells in adult hippocampal neurogenesis.

Drinking alcohol during pregnancy may cause fetal alcohol spectrum disorder. In rats, developmental exposure to ethanol (EtOH) at high doses has shown to induce aberrant neurogenesis in neural progenitor cells (NPCs) during weaning and suppress synaptic plasticity of newborn granule cells after maturation; neuroinflammation was even sustained until the adult stage in the hippocampal dentate gyrus (DG). To investigate whether hippocampal neurogenesis is affected by EtOH exposure in a general toxicity study, EtOH was administered orally to 5-week-old Sprague-Dawley rats at 0%, 10%, and 16% (w/v) in drinking water for 28 days. Exposure to 16% EtOH decreased type-1 neural stem cells (NSCs) and type-2a NPCs in the DG subgranular zone. A reduction in reelin-positive (reelin+) interneurons and an increased number of parvalbumin+ interneurons in the DG hilus, as well as downregulation of Mcm6 and Calb2 in the DG, suggested that self-renewal and proliferation of type-1 NSCs were suppressed. Exposure to 16% EtOH also induced M1-type microglia/peripheral macrophages, and upregulated Il1a and Tnf, suggesting that neuroinflammation might be responsible for the suppressed neurogenesis. In contrast, Drd2 and Tgfb3 upregulation might be ameliorating responses against suppressed neurogenesis. EtOH exposure (16%) also decreased the number of FOS+ granule cells, suggesting that synaptic plasticity was suppressed; concurrent upregulation of glutamate receptor/transporter genes may have occurred as a compensatory response against suppressed synaptic plasticity. Thus, high-dose EtOH exposure in young adult rats disrupted hippocampal neurogenesis differently to exposure during development. However, induction of neuroinflammation and suppressed synaptic plasticity occurred at both EtOH exposure stages.

Ameliorating effect of continuous alpha-glycosyl isoquercitrin treatment starting from late gestation in a rat autism model induced by postnatal injection of lipopolysaccharides.

The present study investigated the role of neuroinflammation and brain oxidative stress induced by neonatal treatment with lipopolysaccharides (LPS) on the development of autism spectrum disorder (ASD)-like behaviors and disruptive hippocampal neurogenesis in rats by exploring the chemopreventive effects of alpha-glycosyl isoquercitrin (AGIQ) as an antioxidant. AGIQ was dietary administered to dams at 0.25% or 0.5% (w/w) from gestational day 18 until postnatal day (PND) 21 on weaning and then to pups until the adult stage on PND 77. The pups were intraperitoneally injected with LPS (1 mg/kg body weight) on PND 3. At PND 6, LPS alone increased Iba1+ and CD68+ cell numbers without changing the CD163+ cell number and strongly upregulated pro-inflammatory cytokine gene expression (Il1a, Il1b, Il6, Nfkb1, and Tnf) in the hippocampus, and increased brain malondialdehyde levels. At PND 10, pups decreased ultrasonic vocalization (USV), suggesting the induction of pro-inflammatory responses and oxidative stress to trigger communicative deficits. By contrast, LPS alone upregulated Nfe2l2 expression at PND 6, increased Iba1+, CD68+, and CD163+ cell numbers, and upregulated Tgfb1 at PND 21, suggesting anti-inflammatory responses until the weaning period. However, LPS alone disrupted hippocampal neurogenesis at weaning and suppressed social interaction parameters and rate of freezing time at fear acquisition and extinction during the adolescent stage. On PND 77, neuroinflammatory responses had mostly disappeared; however, disruptive neurogenesis and fear memory deficits were sustained. AGIQ ameliorated most changes on acute pro-inflammatory responses and oxidative stress at PND 6, and the effects on USVs at PND 10 and neurogenesis and behavioral parameters throughout the adult stage. These results suggested that neonatal LPS treatment induced acute but transient neuroinflammation, triggering the progressive disruption of hippocampal neurogenesis leading to abnormal behaviors in later life. AGIQ treatment was effective for ameliorating LPS-induced progressive changes by critically suppressing initial pro-inflammatory responses and oxidative stress.

Aberrant neurogenesis and late onset suppression of synaptic plasticity as well as sustained neuroinflammation in the hippocampal dentate gyrus after developmental exposure to ethanol in rats.

Drinking alcohol during pregnancy may cause fetal alcohol spectrum disorder. The present study investigated the effects of maternal oral ethanol (EtOH) exposure (0, 10, or 12.5 % in drinking water) from gestational day 6 until day 21 post-delivery (weaning) on postnatal hippocampal neurogenesis at weaning and in adulthood on postnatal day 77 in rat offspring. At weaning, type-3 neural progenitor cells (NPCs) were decreased in the subgranular zone (SGZ), accompanied by Chrnb2 downregulation and Grin2b upregulation in the dentate gyrus (DG). These results suggested suppression of CHRNB2-mediated cholinergic signaling in γ-aminobutyric acid (GABA)ergic interneurons in the DG hilus and increased glutamatergic signaling through the NR2B subtype of N-methyl-d-aspartate (NMDA) receptors, resulting in NPC reduction. In contrast, upregulation of Chrna7 may increase CHRNA7-mediated cholinergic signaling in immature granule cells, and upregulation of Ntrk2 may cause an increase in somatostatin-immunoreactive (+) GABAergic interneurons, suggesting a compensatory response against NPC reduction. Promotion of SGZ cell proliferation increased type-2a NPCs. Moreover, an increase in calbindin-d-29 K+ interneurons and upregulation of Reln, Drd2, Tgfb2, Il18, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor subunit genes might participate in this compensatory response. In adulthood, reduction of FOS+ cells and downregulation of Fos and Arc suggested suppression of granule cell synaptic plasticity, reflecting upregulation of Tnf and downregulation of Cntf, Ntrk2, and AMPA-type glutamate receptor genes. In the DG hilus, gliosis and hyper-ramified microglia, accompanying upregulation of C3, appeared at weaning, suggesting contribution to suppressed synaptic plasticity in adulthood. M1 microglia increased throughout adulthood, suggesting sustained neuroinflammation. These results indicate that maternal EtOH exposure temporarily disrupts hippocampal neurogenesis and later suppresses synaptic plasticity. Induction of neuroinflammation might initially ameliorate neurogenesis (as evident by upregulation of Tgfb2 and Il18) but later suppress synaptic plasticity (as evident by upregulation of C3 at weaning and Tnf in adulthood).

Induction of cellular senescence as a late effect and BDNF-TrkB signaling-mediated ameliorating effect on disruption of hippocampal neurogenesis after developmental exposure to lead acetate in rats.

Lead (Pb) exposure causes cognitive deficits in children. The present study investigated the effect of developmental exposure to Pb acetate (PbAc) on postnatal hippocampal neurogenesis. Pregnant rats were administered drinking water containing 0, 2000, or 4000 ppm PbAc from gestational day 6 until day 21 post-delivery (weaning), and offspring were maintained without PbAc exposure until adulthood on postnatal day (PND) 77. There was a dose-related accumulation of Pb in the offspring brain at weaning, while Pb was mainly excreted in adulthood. In the hippocampus, metallothionein I/II immunoreactive (+) glia were increased through adulthood as a neuroprotective response to accumulated Pb, accompanied by increased astrocyte and microglia numbers in adulthood, suggesting sustained neural damage. Gene expression changes suggested elevated oxidative stress at weaning and suppression of the antioxidant system in adulthood, as well as continued neuroinflammatory responses. At weaning, granule cell apoptosis was increased and numbers of type-3 neural progenitor cells (NPCs) were decreased. By contrast, type-2a and type-2b NPCs were increased, suggesting suppressed differentiation to type-3 NPCs. In adulthood, there were increased numbers of immature granule cells. In the hilus of the dentate gyrus, somatostatin+ interneurons were increased at weaning, while calbindin-D-29K+ interneurons were increased throughout adulthood, suggesting a strengthened interneuron regulatory system against the suppressed differentiation at weaning. In the dentate gyrus, Bdnf, Ntrk2, and Chrna7 gene expression were upregulated and numbers of hilar TrkB+ interneurons increased at weaning. These findings suggest activation of BDNF-TrkB signaling to increase somatostatin+ interneurons and promote cholinergic signaling, thus increasing later production of immature granule cells. In adulthood, Pcna and Apex1 gene expression were downregulated and Chek1 and cyclin-dependent kinase inhibitor expression were upregulated. Furthermore, there was an increase in γ-H2AX+ SGZ cells, suggesting induction of cellular senescence of SGZ cells due to Pb genotoxicity.

A 28-day repeated oral dose toxicity study of enniatin complex in mice.

Enniatins are so-called "emerging mycotoxins" that commonly occur in milligrams per kilogram levels in grains and their derived products, as well as in fish, dried fruits, nuts, spices, cocoa, and coffee. The present study investigated the 28-day repeated oral dose toxicity of enniatin complex in CD1(ICR) mice. Enniatin B, enniatin B1, and enniatin A1 at a ratio of 4:4:1 were administered to male and female mice at doses of 0 (vehicle controls), 0.8, 4, and 20 mg/kg body weight/day. In life parameters did not change during the study period, with the exception of slight reductions in food consumption in male mice administered 4 and 20 mg/kg and in female mice administered 20 mg/kg. Body and organ weights did not change, and no alterations in hematology, blood biochemistry, or histopathology parameters were observed at the end of the administration period. Thus, we determined that the no-observed-adverse-effect level of enniatin complex was 20 mg/kg/day for both sexes under the present experimental conditions.

Strain differences in histopathological features of lymphoid tissues of SD and F344 rats in a T cell-dependent antibody response assay of cyclophosphamide.

When conducting histopathological evaluation of lymphoid tissues, it is necessary to know the variability and strain differences in histological features of different sites of lymphoid tissues. To investigate in detail the variability of lymphoid tissues and strain differences of control rats as well as those of immune reactivity and sensitivity to immunosuppression, we performed a histopathological analysis of various lymphoid tissues in conjunction with the evaluation of immune function in a T cell-dependent antibody response (TDAR) assay with cyclophosphamide (CP) in Sprague Dawley (SD) and F344 rats. Six-week-old male SD and F344 rats were orally treated with CP at 0 (control) or 4 mg/kg/day for 28 days; keyhole limpet hemocyanin (KLH) was introduced intravenously on Days 14 and 23, and the serum concentrations of anti-KLH antibodies were measured. HE staining and immunohistochemistry for T-cell (CD3) and B-cell (CD45RA) markers were performed using tissues from the spleen, thymus, and various lymph nodes. In CP-treated rats of both strains, decreased concentrations of anti-KLH antibodies were observed. Histopathological analysis revealed decreased lymphocytes mainly in the B-cell area, and these changes induced by CP treatment were more prominent in the F344 rats than in the SD rats. The present study also demonstrated that some of the lymphoid tissues of the control F344 rats were less developed than those of the control SD rats, suggesting that F344 rats might be easily affected by CP-induced immunosuppression. This information concerning rat strain differences in lymphoid tissues will be useful in histopathological evaluation for drug-induced immunotoxicity.

Influence of the estrus cycle on the evaluation of a vaginal irritation study in intact and ovariectomized rats.

When conducting vaginal irritation studies, ovariectomized rats or rabbits are typically used according to practical reports. In the present study, we evaluated the influence of the estrus cycle in a vaginal irritation study using intact rats and ovariectomized rats, which exhibit a late diestrus-like condition, to determine whether intact rats can be useful for evaluating vaginal irritancy. Rats were divided into 4 groups: proestrus, estrus, and metestrus or diestrus in intact rats and ovariectomized rats. All the rats in each group were treated with a vehicle or sodium dodecyl sulfate, as the irritant, in single-dose and 4-day repeat-dose vaginal irritation studies. Each rat's vagina was examined histopathologically, and the irritation score was calculated using a semiquantitative scoring system. In the single-dose study, the irritation scores for the proestrus or ovariectomized groups treated with sodium dodecyl sulfate were higher than those of the estrus group or metestrus or diestrus group. In the 4-day repeat-dose study, a significant histopathological difference was not found among the intact rats (proestrus, estrus, and metestrus or diestrus groups), and the irritation score range of the intact rats was similar to that of the ovariectomized rats, though the mean score of the intact rats was slightly lower than that of the ovariectomized rats. These results suggest that intact rats might be well suited for 4-day vaginal irritation studies and useful for evaluating vaginal irritancy using not only the mean score, but also individual irritation score ranges, whereas the estrus cycle would need to be identified in single-dose vaginal irritation studies.

Effects of reduced food intake for 4 weeks on physiological parameters in toxicity studies in dogs.

This study sought to clarify the effects of reduced feeding on physiological parameters in dogs to enable appropriate evaluations of the safety and toxicity of test compounds. We measured alkaline phosphatase isozymes and the circulating blood volume, as well as clinical signs, body weight, hematology, blood chemistry, electrocardiography, organ weight, and histopathology, in male beagle dogs fed a diet consisting of 300 g/day or 150 g/day for 4 weeks. There were no abnormal clinical signs in any of the dogs. In the 150-g/day feeding group, a decreased alkaline phosphatase 3 suggesting effects on the bone and a decreased circulating blood volume associated with body weight loss were observed. Additionally, the following changes were also observed in the 150-g/day group: a decrease in body weight; hematologic changes including decreases in white blood cells, neutrophils, red blood cells, hemoglobin, hematocrit and reticulocytes; blood chemical changes including decreases in aspartate aminotransferase, lactate dehydrogenase and calcium and an increase in the creatinine at week 1 or thereafter; electrocardiographic changes including a decrease in the heart rate, a prolonged QRS duration and the occurrence of a second-degree atrioventricular block at week 3 or thereafter; and pathological changes including decreases in the weights of the liver and thymus, a decrease in hepatocyte rarefaction, and thymic atrophy. These results provide useful information for assessing the safety of compounds in toxicological studies, enabling direct treatment effects and secondary changes caused by decreased food intake to be distinguished.

Reversible aberration of neurogenesis targeting late-stage progenitor cells in the hippocampal dentate gyrus of rat offspring after maternal exposure to acrylamide.

We have recently shown that maternal exposure to acrylamide (AA) impaired neurogenesis in rat offspring measured by the increase in interneurons producing reelin, a molecule regulating migration and correct positioning of developing neurons, in the hippocampal dentate gyrus. To clarify the cellular target of AA on hippocampal neurogenesis and its reversibility after maternal exposure, pregnant Sprague-Dawley rats were given drinking water containing AA at 0, 4, 20, 100 ppm on day 10 of pregnancy through day 21 after delivery on weaning. Male offspring were examined immunohistochemically on postnatal day (PND) 21 and PND 77. For comparison, male pups of direct AA-injection control during lactation (50 mg/kg body weight, intraperitoneally, 3 times/week) were also examined. On PND 21, maternal AA-exposure decreased progenitor cell proliferation in the subgranular zone (SGZ) from 20 ppm accompanied with increased density of reelin-producing interneurons and NeuN-expressing mature neurons within the hilus at 100 ppm, similar to the direct AA-injection control. In the SGZ examined at 100 ppm, cellular populations immunoexpressing doublecortin or dihydropyrimidinase-like 3, suggesting postmitotic immature granule cells, were decreased. On PND 77, the SGZ cell proliferation and reelin-producing interneuron density recovered, while the hilar mature neurons sustained to increase from 20 ppm, similar to the direct AA-injection control. Thus, developmental exposure to AA reversibly affects hippocampal neurogenesis targeting the proliferation of type-3 progenitor cells resulting in a decrease in immature granule cells in rats. A sustained increase in hilar mature neurons could be the signature of the developmental effect of AA.

Neuroendocrine carcinoma of the apocrine glands of the anal sac in a dog.

A perianal subcutaneous tumor involving the anal sac developed in an 8-year-old male mixed Labrador Retriever dog. Histologically, this tumor showed typical features of the solid-type carcinoma of the apocrine glands of the anal sac. However, neoplastic cells were immunoreactive for cytokeratin 8, chromogranin A, vasoactive intestinal peptide, neuron-specific enolase, and synaptophysin, and negative for S-100 protein, α-smooth muscle actin, vimentin, glucagon, insulin, somatostatin, carcinoembryonic antigen, serotonin, and parathyroid hormone-related protein. Considering the distribution of chromogranin A-positive cells within the anal sac apocrine glands, this tumor was diagnosed as neuroendocrine carcinoma originating from the apocrine glands of the anal sac.

Suppressive effect of enzymatically modified isoquercitrin on phenobarbital-induced liver tumor promotion in rats.

To investigate the effect of enzymatically modified isoquercitrin (EMIQ) on hepatocellular tumor promotion induced by phenobarbital (PB), male rats were administered a single intraperitoneal injection of 200 mg/kg N-diethylnitrosamine (DEN) and then fed with a diet containing PB (500 ppm) for 8 weeks, with or without EMIQ (2,000 ppm) in the drinking water. One week after PB administration, rats underwent a two-thirds partial hepatectomy. The PB-induced increase in the number and area of glutathione S-transferase placental form-positive foci and the proliferating cell nuclear antigen-positive ratio was significantly suppressed by EMIQ. Real-time reverse transcription-polymerase chain reaction analysis revealed increases in mRNA expression levels of Cyp2b2 and Mrp2 in the DEN-PB and DEN-PB-EMIQ groups compared with the DEN-alone group, while the level of Mrp2 decreased in the DEN-PB-EMIQ group compared with the DEN-PB group. There were no significant changes in microsomal reactive oxygen species (ROS) production and oxidative stress markers between the DEN-PB and DEN-PB-EMIQ groups. Immunohistochemically, the constitutive active/androstane receptor (CAR) in the DEN-PB group was clearly localized in the nuclei, but its immunoreactive intensity was decreased in the DEN-PB-EMIQ group. These results indicate that EMIQ suppressed the liver tumor-promoting activity of PB by inhibiting nuclear translocation of CAR, and not by suppression of oxidative stress.

Disruptive neuronal development by acrylamide in the hippocampal dentate hilus after developmental exposure in rats.

To examine whether developmental exposure to acrylamide (AA) impairs neuronal development, pregnant Sprague-Dawley rats were treated with AA at 0, 25, 50 or 100 ppm in drinking water from gestational day 6 until weaning on postnatal day 21. Offspring were immunohistochemically examined at the end of exposure. We investigated the expression of Reelin (a molecule regulating neuronal migration and positioning) in the hilus of the hippocampal dentate gyrus. As a positive control for direct exposure, AA (50 mg/kg body weight) was administered to pups by intraperitoneal injection 3 times per week during the lactation period. As well as pups directly injected with AA, maternally exposed offspring decreased body weight at 100 ppm; increased dose-dependently the number of Reelin-immunoreactive cells (from 25 ppm AA) and glutamic acid decarboxylase 67-immunoreactive cells (from 50 ppm AA), confirming an increase in γ-aminobutyric acid-ergic interneurons. We also noted decreased apoptosis in the neuroblast-producing subgranular zone of the dentate gyrus of maternally exposed pups at 100 ppm, as well as in directly AA-injected pups. These results suggest that a compensatory regulatory mechanism exists to correct impaired neurogenesis and mismigration caused by maternal exposure to AA during neuronal development. The lowest-observed-adverse-effect level of AA was determined to be 25 ppm (3.72 mg/kg body weight/day).

No effect of sustained systemic growth retardation on the distribution of Reelin-expressing interneurons in the neuron-producing hippocampal dentate gyrus in rats.

Reelin signaling plays a role in neuronal migration and positioning during brain development. To clarify the effect of systemic growth retardation on the distribution of Reelin-expressing interneurons in the hilus of the hippocampal dentate gyrus, pregnant rats were fed a synthetic diet with either a normal (20% casein) or low (10% casein) protein concentration from gestational day 10 to postnatal day (PND) 21 at weaning. Male offspring were immunohistochemically examined at PND 21 and on PND 77. Protein-restricted offspring displayed systemic growth retardation through PND 77 and had decreased absolute brain weights and an increased number of external granular cells in the cerebellar cortex, suggestive of retarded brain growth at weaning. However, maternal protein restriction did not change the cellular distribution of immunoreactivity for Reelin, Calbindin-D-28K, or glutamic acid decarboxylase 67 or of NeuN-positive postmitotic neurons in the dentate hilus either at PND 21 or PND 77, which suggests that the population of γ-aminobutyric acid-ergic interneurons involving synthesis of Reelin was not affected. Furthermore, as well as the distribution of hilar neurons expressing neurogenesis-related FoxG1, cell proliferation and apoptosis in the subgranular zone were unaffected through PND 77. These results suggest that systemic growth retardation caused by maternal protein restriction does not affect neuronal migration and postnatal neurogenesis of the dentate gyrus resulting in unaltered distribution of Reelin-synthesizing interneurons.

A mixed apocrine gland tumor with metastases to the bone and bone marrow in a miniature poodle.

A 10-year-old female miniature poodle had a mass in its carpal joint of the left forelimb. The tumor was divided into small multiple lobules by delicate connective tissues, and necroses were found in some of the central lobules. In some connective stromal areas, chondroid and osteoid tissues were formed. The tumor cells were similar to the structure of apocrine gland epithelial cells with apical blebs resembling apocrine secretion and eosinophilic secretary materials within the luminal space, and spindle cells were sometimes found in the basal area of the glandular structure. In some areas, tumor cells invaded in the blood vessels, bone and bone marrow. Immunohistochemically, the tumor cells forming tubulo-acinar to solid structures were intensely positive for cytokeratin and keratin K8/K18, and the spindle cells were positive for vimentin and alpha-smooth muscle actin. This case was diagnosed as a malignant mixed apocrine gland tumor with metastases to the bone and bone marrow.

A neuroendocrine carcinoma of undetermined origin in a dog.

In this report, we describe a case of neuroendocrine carcinoma of undetermined origin in a dog. Necropsy revealed scattered small neoplastic nodules in the bilateral lungs and a small nodule in the parapancreatic lymph node. Histopathologically, both pulmonary and lymph nodal nodules showed a similar histologic pattern, with neoplastic cells being arranged in diffusely proliferating sheet-like cellular nests separated by variable amounts of fibrous septa, sometimes forming rosettes and duct-like structures. Scattered small necrotic foci and invasion to fibrous septa were typically observed. Neoplastic cells showed round to oval-shaped nuclei with prominent nucleoli and abundant eosinophilic cytoplasm that were positive for Grimelius' silver impregnation staining and immunostaining with cytokeratin, synaptophysin, vasoactive intestinal peptide and chromogranin A, indicative of the development of a neuroendocrine carcinoma. However, judging from the distribution of tumors lacking the portion suggestive of the primary site in any organ examined, as well as no further indication of differentiation potential of neoplastic cells, this tumor has so far been diagnosed as neuroendocrine carcinoma of undetermined origin.