Distinct placental malaria pathology caused by different Plasmodium berghei lines that fail to induce cerebral malaria in the C57BL/6 mouse

Background: Placental malaria (PM) is one major feature of malaria during pregnancy. A murine model of experimental PM using BALB/c mice infected with Plasmodium berghei ANKA was recently established, but there is need for additional PM models with different parasite/host combinations that allow to interrogate the involvement of specific host genetic factors in the placental inflammatory response to Plasmodium infection. Methods: A mid-term infection protocol was used to test PM induction by three P. berghei parasite lines, derived from the K173, NK65 and ANKA strains of P. berghei that fail to induce experimental cerebral malaria (ECM) in the susceptible C57BL/6 mice. Parasitaemia course, pregnancy outcome and placenta pathology induced by the three parasite lines were compared. Results: The three P. berghei lines were able to evoke severe PM pathology and poor pregnancy outcome features. The results indicate that parasite components required to induce PM are distinct from ECM. Nevertheless, infection with parasites of the ANKA Delta pm4 line, which lack expression of plasmepsin 4, displayed milder disease phenotypes associated with a strong innate immune response as compared to infections with NK65 and K173 parasites. Conclusions: Infection of pregnant C57BL/6 females with K173, NK65 and ANKA Delta pm4 P. berghei parasites provide experimental systems to identify host molecular components involved in PM pathogenesis mechanisms.

Cocaine effects on mouse incentive-learning and human addiction are linked to alpha 2 subunit-containing GABA(A) receptors

Because GABA(A) receptors containing alpha 2 subunits are highly represented in areas of the brain, such as nucleus accumbens (NAcc), frontal cortex, and amygdala, regions intimately involved in signaling motivation and reward, we hypothesized that manipulations of this receptor subtype would influence processing of rewards. Voltage-clamp recordings from NAcc medium spiny neurons of mice with alpha 2 gene deletion showed reduced synaptic GABA(A) receptor-mediated responses. Behaviorally, the deletion abolished cocaine`s ability to potentiate behaviors conditioned to rewards (conditioned reinforcement), and to support behavioral sensitization. In mice with a point mutation in the benzodiazepine binding pocket of alpha 2-GABA(A) receptors (alpha 2H101R), GABAergic neurotransmission in medium spiny neurons was identical to that of WT (i.e., the mutation was silent), but importantly, receptor function was now facilitated by the atypical benzodiazepine Ro 15-4513 (ethyl 8-amido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5-a] [1,4] benzodiazepine-3-carboxylate). In alpha 2H101R, but not WT mice, Ro 15-4513 administered directly into the NAcc-stimulated locomotor activity, and when given systemically and repeatedly, induced behavioral sensitization. These data indicate that activation of alpha 2-GABA(A) receptors (most likely in NAcc) is both necessary and sufficient for behavioral sensitization. Consistent with a role of these receptors in addiction, we found specific markers and haplotypes of the GABRA2 gene to be associated with human cocaine addiction.

Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow

Under homeostatic conditions, a proportion of senescent CXCR4(hi) neutrophils home from the circulation back to the bone marrow, where they are phagocytosed by bone marrow macrophages. In this study, we have identified an unexpected role for the anti-inflammatory molecule annexin A1 (AnxA1) as a critical regulator of this process. We first observed that AnxA1(-/-) mice have significantly increased neutrophil numbers in their bone marrow while having normal levels of GM and G colony-forming units, monocytes, and macrophages. Although AnxA1(-/-) mice have more neutrophils in the bone marrow, a greater proportion of these cells are senescent, as determined by their higher levels of CXCR4 expression and annexin V binding. Consequently, bone marrow neutrophils from AnxA1(-/-) mice exhibit a reduced migratory capacity in vitro. Studies conducted in vitro also show that expression of AnxA1 is required for bone marrow macrophages, but not peritoneal macrophages, to phagocytose apoptotic neutrophils. Moreover, in vivo experiments indicate a defect in clearance of wild-type neutrophils in the bone marrow of AnxA1(-/-) mice. Thus, we conclude that expression of AnxA1 by resident macrophages is a critical determinant for neutrophil clearance in the bone marrow.-Dalli, J., Jones, C. P., Cavalcanti, D. M., Farsky, S. H., Perretti, M., Rankin, S. M. Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow. FASEB J. 26, 387-396 (2012). www.fasebj.org

Clinico-genetic aspects of a pediatric non-neurofibromatosis type 1 malignant triton tumor with loss of chromosome X

Malignant triton tumor (MTT) is an aggressive peripheral nerve sheath tumor with rhabdomyoblastic differentiation. Less than 100 cases have been described, being mostly male children with type 1 neurofibromatosis. We report a 6-year-old female with MTT and no diagnostic criteria for neurofibromatosis type 1. Cytogenetic analysis showed a 46,X,-X[4]/46,XX[16] karyotype. She underwent a transfemoral amputation and chemotherapy and is free of disease 15 months after diagnosis. The few cytogenetic studies of MTT described in the literature have been inconclusive. Further cytogenetic analyses are needed to understand the role of chromosome X monosomy in the pathogenesis of this rare tumor. Pediatr Blood Cancer 2012; 59: 13201323. (C) 2012 Wiley Periodicals, Inc.

Intermittent hypoxia inhibits clearance of triglyceride-rich lipoproteins and inactivates adipose lipoprotein lipase in a mouse model of sleep apnoea

Delayed lipoprotein clearance is associated with atherosclerosis. This study examined whether chronic intermittent hypoxia (CIH), a hallmark of obstructive sleep apnoea (OSA), can lead to hyperlipidaemia by inhibiting clearance of triglyceride rich lipoproteins (TRLP). Male C57BL/6J mice on high-cholesterol diet were exposed to 4 weeks of CIH or chronic intermittent air (control). FIO2 was decreased to 6.5 once per minute during the 12 h light phase in the CIH group. After the exposure, we measured fasting lipid profile. TRLP clearance was assessed by oral gavage of retinyl palmitate followed by serum retinyl esters (REs) measurements at 0, 1, 2, 4, 10, and 24 h. Activity of lipoprotein lipase (LpL), a key enzyme of lipoprotein clearance, and levels of angiopoietin-like protein 4 (Angptl4), a potent inhibitor of the LpL activity, were determined in the epididymal fat pads, skeletal muscles, and heart. Chronic intermittent hypoxia induced significant increases in levels of total cholesterol and triglycerides, which occurred in TRLP and LDL fractions (P 0.05 for each comparison). Compared with control mice, animals exposed to CIH showed increases in REs throughout first 10 h after oral gavage of retinyl palmitate (P 0.05), indicating that CIH inhibited TRLP clearance. CIH induced a 5-fold decrease in LpL activity (P 0.01) and an 80 increase in Angptl4 mRNA and protein levels in the epididymal fat, but not in the skeletal muscle or heart. CIH decreases TRLP clearance and inhibits LpL activity in adipose tissue, which may contribute to atherogenesis observed in OSA.

Co-existence of t(6;13)(p21;q14.1) and trisomy 12 in chronic lymphocytic leukemia

We report a case of a 57-year-old man diagnosed with chronic lymphocytic leukemia (CLL) and presence of a rare t(6;13)(p21;q14.1) in association with an extra copy of chromosome 12. Classical cytogenetic analysis using the immunostimulatory combination of DSP30 and IL-2 showed the karyotype 47,XY,t(6;13)(p21;q14.1), +12 in 75% of the metaphase cells. Spectral karyotype analysis (SKY) confirmed the abnormality previously seen by G-banding. Additionally, interphase fluorescence in situ hybridization using an LSI CEP 12 probe performed on peripheral blood cells without any stimulant agent showed trisomy of chromosome 12 in 67% of analyzed cells (134/200). To the best of our knowledge, the association of t(6;13)(p21;q14.1) and +12 in CLL has never been described. The prognostic significance of these new findings in CLL remains to be elucidated. However, the patient has been followed up since 2009 without any therapeutic intervention and has so far remained stable.

Antiproliferative Effects of Fluoxetine on Colon Cancer Cells and in a Colonic Carcinogen Mouse Model

The antidepressant fluoxetine has been under discussion because of its potential influence on cancer risk. It was found to inhibit the development of carcinogen-induced preneoplastic lesions in colon tissue, but the mechanisms of action are not well understood. Therefore, we investigated anti-proliferative effects, and used HT29 colon tumor cells in vitro, as well as C57BL/6 mice exposed to intra-rectal treatment with the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as models. Fluoxetine increased the percentage of HT29 cells in the G(0)/G(1) phase of cell-cycle, and the expression of p27 protein. This was not related to an induction of apoptosis, reactive oxygen species or DNA damage. In vivo, fluoxetine reduced the development of MNNG-induced dysplasia and vascularization-related dysplasia in colon tissue, which was analyzed by histopathological techniques. An anti-proliferative potential of fluoxetine was observed in epithelial and stromal areas. It was accompanied by a reduction of VEGF expression and of the number of cells with angiogenic potential, such as CD133, CD34, and CD31-positive cell clusters. Taken together, our findings suggest that fluoxetine treatment targets steps of early colon carcinogenesis. This confirms its protective potential, explaining at least partially the lower colon cancer risk under antidepressant therapy.

alpha-6 Integrin Expression in Bovine Spermatogonial Cells Purified by Discontinuous Percoll Density Gradient

The study of spermatogonial stem cells (SSCs) provides a model to better understand adult stem cell biology. Besides the biomedical potential to perform studies of infertility in many species, SSCs hold a promising application at animal transgenesis. Because stem cells are thought to be associated with basement membranes, expression of alpha-6 integrin has been investigated as a marker of type A spermatogonial cells, which are considered SSCs because of their undifferentiated status and self-renewal ability. In this manner, the aim of this study was to isolate type A SSCs from adult bulls by a two-step enzymatic procedure followed by a discontinuous Percoll density gradient purification and verify the expression of alpha-6 integrin by flow cytometry and real-time RT-PCR before and after Percoll purification. Spermatogonial cells were successfully obtained using the two-step enzymatic digestion. An average of 1 x 10(5) viable cells per gram of testis was isolated. However, the discontinuous Percoll did not purify isolated cells regarding alpha-6 integrin expression. Flow cytometry analysis demonstrated no differences in the alpha-6 integrin expression between cell samples before and after Percoll purification (p = 0.5636). The same was observed in the real-time PCR analysis (p > 0.05). In addition to alpha-6 integrin, the expression of GFR alpha-1 and PGP9.5, known bovine SSCs markers, was detected in all samples studied. Considering that Percoll can reduce cell viability, it is possible to conclude that Percoll density gradient is not suitable to purify bovine SSC, according to alpha-6 integrin expression.

Expression of the P2X(2) receptor in different classes of ileum myenteric neurons in the female obese ob/ob mouse

AIM: To examine whether the ob/ob mouse model of obesity is accompanied by enteric nervous system abnormalities such as altered motility. METHODS: The study examined the distribution of the P2X(2) receptor (P2X(2)R) in myenteric neurons of female ob/ob mice. Specifically, we used immunohistochemistry to analyze the co-expression of the P2X(2)R with neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and calretinin (CalR) in neurons of the small intestine myenteric plexus in ob/ob and control female mice. In these sections, we used scanning confocal microscopy to analyze the co-localization of these markers as well as the neuronal density (cm(2)) and area profile (mu m(2)) of P2X(2)R-positive neurons. In addition, enteric neurons were labeled using the nicotinamide adenine dinucleotide (NADH) diaphorase method and analyzed with light microscopy as an alternate means by which to analyze neuronal density and area. RESULTS: In the present study, we observed a 29.6% increase in the body weight of the ob/ob animals (OG) compared to the control group (CG). In addition, the average small intestine area was increased by approximately 29.6% in the OG compared to the CG. Immunoreactivity (IR) for the P2X(2)R, nNOS, ChAT and CaIR was detectable in the myenteric plexus, as well as in the smooth muscle, in both groups. This IR appeared to be mainly cytoplasmic and was also associated with the cell membrane of the myenteric plexus neurons, where it outlined the neuronal cell bodies and their processes. P2X(2)R-IR was observed to co-localize 100% with that for nNOS, ChAT and CaIR in neurons of both groups. In the ob/ob group, however, we observed that the neuronal density (neuron/cm(2)) of P2X(2)R-IR cells was increased by 62% compared to CG, while that of NOS-IR and ChAT-IR neurons was reduced by 49% and 57%, respectively, compared to control mice. The neuronal density of CaIR-IR neurons was not different between the groups. Morphometric studies further demonstrated that the cell body profile area (mu m(2)) of nNOS-IR, ChAT-IR and CaIR-IR neurons was increased by 34%, 20% and 55%, respectively, in the OG compared to controls. Staining for NADH diaphorase activity is widely used to detect alterations in the enteric nervous system; however, our qualitative examination of NADH-diaphorase positive neurons in the nnyenteric ganglia revealed an overall similarity between the two groups. CONCLUSION: We demonstrate increases in P2X(2)R expression and alterations in nNOS, ChAT and CaIR IR in ileal myenteric neurons of female ob/ob mice compared to wild-type controls. (c) 2012 Baishideng. All rights reserved.

Chromosome painting in three-toed sloths: a cytogenetic signature and ancestral karyotype for Xenarthra

Background: Xenarthra (sloths, armadillos and anteaters) represent one of four currently recognized Eutherian mammal supraorders. Some phylogenomic studies point to the possibility of Xenarthra being at the base of the Eutherian tree, together or not with the supraorder Afrotheria. We performed painting with human autosomes and X-chromosome specific probes on metaphases of two three-toed sloths: Bradypus torquatus and B. variegatus. These species represent the fourth of the five extant Xenarthra families to be studied with this approach. Results: Eleven human chromosomes were conserved as one block in both B. torquatus and B. variegatus: (HSA 5, 6, 9, 11, 13, 14, 15, 17, 18, 20, 21 and the X chromosome). B. torquatus, three additional human chromosomes were conserved intact (HSA 1, 3 and 4). The remaining human chromosomes were represented by two or three segments on each sloth. Seven associations between human chromosomes were detected in the karyotypes of both B. torquatus and B. variegatus: HSA 3/21, 4/8, 7/10, 7/16, 12/22, 14/15 and 17/19. The ancestral Eutherian association 16/19 was not detected in the Bradypus species. Conclusions: Our results together with previous reports enabled us to propose a hypothetical ancestral Xenarthran karyotype with 48 chromosomes that would differ from the proposed ancestral Eutherian karyotype by the presence of the association HSA 7/10 and by the split of HSA 8 into three blocks, instead of the two found in the Eutherian ancestor. These same chromosome features point to the monophyly of Xenarthra, making this the second supraorder of placental mammals to have a chromosome signature supporting its monophyly.

Cytogenomic characterization of an unexpected 17.6 Mb 9p deletion associated to a 14.8 Mb 20p duplication in a dysmorphic patient with multiple congenital anomalies presenting a normal G-banding karyotype

We describe a female patient with developmental delay, dysmorphic features and multiple congenital anomalies who presented a normal G-banded karyotype at the 550-band resolution. Array and multiplex-ligation probe amplification (MLPA) techniques identified an unexpected large unbalanced genomic aberration: a 17.6 Mb deletion of 9p associated to a 14.8 Mb duplication of 20p. The deleted 9p genes, especially CER1 and FREM1, seem to be more relevant to the phenotype than the duplicated 20p genes. This study also shows the relevance of using molecular techniques to make an accurate diagnosis in patients with dysmorphic features and multiple anomalies suggestive of chromosome aberration, even if on G-banding their karyotype appears to be normal. Fluorescence in situ hybridization (FISH) was necessary to identify a masked balanced translocation in the patient's mother, indicating the importance of associating cytogenetic and molecular techniques in clinical genetics, given the implications for patient management and genetic counseling. (C) 2012 Elsevier B.V. All rights reserved.

Delineation of vertebral area on the coronal plane using three-dimensional ultrasonography advanced volume contrast imaging (VCI) Omni view: intrarater reliability and agreement using standard mouse, high definition mouse, and pen-tablet

Objective: To assess the fetal lumbosacral spine by three-dimensional (3D) ultrasonography using volume contrast imaging (VCI) omni view method and compare reproducibility and agreement between three different measurement techniques: standard mouse, high definition mouse and pen-tablet. Methods: A comparative and prospective study with 40 pregnant women between 20 and 34+6 weeks was realized. 3D volume datasets of the fetal spine were acquired using a convex transabdominal transducer. Starting scan plane was the coronal section of fetal lumbosacral spine by VCI-C function. Omni view manual trace was selected and a parallel plane of fetal spine was drawn including interest region. Intraclass correlation coefficient (ICC) was used for reproducibility analysis. The relative difference between three used techniques was compared by chi-square test and Fischer test. Results: Pen-tablet showed better reliability (ICC = 0.987). In the relative proportion of differences, this was significantly higher for the pen-tablet (82.14%; p < 0.01). In paired comparison, the relative difference was significantly greater for the pen-tablet (p < 0.01). Conclusion: The pen-tablet showed to be the most reproductive and concordant method in the measurement of body vertebral area of fetal lumbosacral spine by 3D ultrasonography using the VCI.

Multiple Intravenous Administrations of Human Umbilical Cord Blood Cells Benefit in a Mouse Model of ALS

Background: A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS) is the use of cell-based therapies that can protect motor neurons and thereby retard disease progression. We recently showed that a single large dose (25x10(6) cells) of mononuclear cells from human umbilical cord blood (MNC hUCB) administered intravenously to pre-symptomatic G93A SOD1 mice is optimal in delaying disease progression and increasing lifespan. However, this single high cell dose is impractical for clinical use. The aim of the present pre-clinical translation study was therefore to evaluate the effects of multiple low dose systemic injections of MNC hUCB cell into G93A SOD1 mice at different disease stages. Methodology/Principal Findings: Mice received weekly intravenous injections of MNC hUCB or media. Symptomatic mice received 10(6) or 2.5x10(6) cells from 13 weeks of age. A third, pre-symptomatic, group received 10(6) cells from 9 weeks of age. Control groups were media-injected G93A and mice carrying the normal hSOD1 gene. Motor function tests and various assays determined cell effects. Administered cell distribution, motor neuron counts, and glial cell densities were analyzed in mouse spinal cords. Results showed that mice receiving 10(6) cells pre-symptomatically or 2.5x10(6) cells symptomatically significantly delayed functional deterioration, increased lifespan and had higher motor neuron counts than media mice. Astrocytes and microglia were significantly reduced in all cell-treated groups. Conclusions/Significance: These results demonstrate that multiple injections of MNC hUCB cells, even beginning at the symptomatic disease stage, could benefit disease outcomes by protecting motor neurons from inflammatory effectors. This multiple cell infusion approach may promote future clinical studies.

Plasma corticosterone levels in mouse models of pain

Background: Pain markedly activates the hypothalamic-pituitary-adrenal (HPA) axis and increases plasma corticosterone release interfering significantly with nociceptive behaviour as well as the mechanism of action of analgesic drugs. Aims/Methods: In the present study, we monitored the time course of circulating corticosterone in two mouse strains (C57Bl/6 and Balb/C) under different pain models. In addition, the stress response was investigated following animal handling, intrathecal (i.t.) manipulation and habituation to environmental conditions commonly used in nociceptive experimental assays. We also examined the influence of within-cage order of testing on plasma corticosterone. Results: Subcutaneous injection of capsaicin precipitated a prompt stress response whereas carrageenan and complete Freund's adjuvant induced an increased corticosterone release around the third hour post-injection. However, carrageenan induced a longer increased corticosterone in C57Bl/6 mice. In partial sciatic nerve ligation, neuropathic pain model corticosterone increased only in the first days whereas mechanical hypersensitivity remained much longer. Animal handling also represents an important stressor whereas the i.t. injection per se does not exacerbate the handling-induced stress response. Moreover, the order of testing animals from the same cage does not interfere with plasma corticosterone levels in the intrathecal procedure. Animal habituation to the testing apparatus also does not reduce the immediate corticosterone increase as compared with non-habituated mice. Conclusion: Our data indicate that HPA axis activation in acute and chronic pain models is time dependent and may be dissociated from evoked hyperalgesia. Therefore, HPA-axis activation represents an important variable to be considered when designing experimental assays of persistent pain as well as for interpretation of data.

Phylogeographic Structure and Karyotypic Diversity of the Brazilian Shrew Mouse (Blarinomys breviceps, Sigmodontinae) in the Atlantic Forest

Blarinomys breviceps possesses cryptic and burrowing habits with poorly documented genetics and life history traits. Due to its rarity, only a few specimens and DNA sequences have been deposited in collections worldwide. Here, we present the most comprehensive cytogenetic and molecular characterization of this rare genus. Phylogenetic analyses based on partial cytochrome b sequences were performed, attempting to establish the relationships among individuals with distinct karyotypes along the geographic distribution of the genus in the Atlantic Forest. Classical and molecular cytogenetics, using banding patterns and FISH of telomeric and whole chromosome X-specific painting probes (obtained from the Akodontini Akodon cursor) were used to characterize and compare the chromosomal complements. Molecular phylogenetic analyses recovered 2 main geographically structured clades, northeastern and southeastern with pair-wise sequence divergences among specimens varying between 4.9 and 8.4%. Eight distinct karyomorphs are described: (A) 2n = 52 (50A, XX), (B) 2n = 52 (48A, XY+2Bs), (C) 2n = 45 (42A, XY+1B), (D) 2n = 43 (37A, XX+4Bs), (E) 2n = 37 (34A, XY+1B), (F) 2n = 34 (32A, XX), (G) 2n = 31 (27A, XX+2Bs), (H) 2n = 28 (26A, XY), all with the same number of autosomal arms (FNA = 50). Variation of 0-4 supernumerary chromosomes (Bs) presenting heterogeneity in morphology and distribution of interstitial telomeric sequences (ITSs) is reported. ITSs are also found in some metacentric autosomes. The phylogeographic separation between 2 major lineages with high levels of genetic divergence, and the wide karyotypic diversity indicate that B. breviceps is a diverse group that warrants taxonomic re-evaluation. Copyright (C) 2012 S. Karger AG, Basel