Neurovirulence of the UC-2 and UC-8 Strains of Bluetongue Virus Serotype 11 in Newborn Mice

1987 ◽  
Vol 24 (5) ◽  
pp. 404-410 ◽  
Author(s):  
A. S. Waldvogel ◽  
C. A. Anderson ◽  
R. J. Higgins ◽  
B. I. Osburn
Keyword(s):  
Bluetongue Virus ◽  
Immunofluorescent Staining ◽  
Target Cells ◽  
Neural Cells ◽  
Inoculation Site ◽  
Virus Serotype ◽  
The Difference ◽  
The Brain ◽  
Subcutaneous Inoculation

In vivo and in vitro experiments were done to investigate whether the difference in neurovirulence between the two strains of bluetongue virus 11, UC-2 and UC-8, is based on a different capability to gain access to the brain from the subcutaneous inoculation site or on a different tropism for neural cells. In newborn Balb/c mice subcutaneous inoculation of UC-8 at doses between 10−0.2 plaque forming units (PFU) and 104.8 PFU caused a severe necrotizing encephalitis whereas UC-2 at doses of up to 104.4 PFU did not affect newborn Balb/c mice. However, intracranial inoculation of 102.4 PFU of either virus strain produced severe necrotizing encephalitis. In vitro both virus strains infected dissociated brain cell cultures similarly. Double labelling immunofluorescent staining with markers specific for neural cells did not reveal differences in the target cells for the two viruses. The difference in neurovirulence between UC-2 and UC-8, therefore, appears to be determined by the ability of UC-8 to infect the brain from a subcutaneous inoculation site.

192 SUSCEPTIBILITY OF BOVINE-HATCHED BLASTOCYSTS TO BLUETONGUE VIRUS SEROTYPE 8 INFECTION

2010 ◽  
Vol 22 (1) ◽  
pp. 254
Author(s):  
L. Vandaele ◽  
W. Wesselingh ◽  
K. De Clercq ◽  
H. Nauwynck ◽  
A. Van Soom
Keyword(s):  
Virus Infection ◽  
Bluetongue Virus ◽  
Calf Serum ◽  
Virus Antigen ◽  
Emerging Diseases ◽  
Immunofluorescent Staining ◽  
Research Centre ◽  
Virus Serotype ◽  
Long Time

In 2006 and 2007, Bluetongue virus serotype 8 (BTV-8) caused devastating outbreaks in Northern Europe; the outbreaks were controlled in 2008 and 2009 by an international vaccination policy. Remarkably, BTV-8 differs from other serotypes in that it spread transplacentally (De Clercq K et al. 2008 Transboundary and Emerging Diseases 55, 352-359). Apart from the transplacental spreading, a significant increase in the incidence of abortions was reported in Belgium (Meroc E et al. 2009 Transboundary and Emerging Diseases 56, 39-48). The aim of the present study was to investigate the susceptibility of bovine-hatched, in vitro-produced blastocysts to BTV-8. A total of 1390 immature bovine oocytes were matured and fertilized in vitro. Presumed zygotes (n = 1148) were denuded 24 h post-insemination and cultured in 50-μL droplets of modified synthetic oviduct fluid (SOF) medium with 10% fetal calf serum (tested negative for BTV antibodies) at 39.0°C in 5% CO2, 5% O2, and 90% N2. At 7 days post-insemination (dpi), blastocysts were grouped to enhance hatching. For virus incubation, BTV-8 Bel 2006/2 from Veterinary and Agrochemical Research Centre (VAR, Brussels, Belgium) was used. At 8.5 dpi, hatched embryos were placed in 800μL of minimum essential medium (MEM) containing 103.8 50% tissue culture infectious doses (TCID50) of BTV-8 and incubated for 1 h at 39°C in an atmosphere of 5% CO2 in air. At the same time, 2 groups of hatched control embryos were incubated under the same circumstances in 800 μL of SOF and 800 μL of MEM, respectively. After infection, all embryos were washed according to IETS guidelines with the exception that they were not zona pellucida intact and cultured in new SOF. At 48, 60, 72, and 96 h post-infection (hpi), one-fourth of the embryos of each group were fixed in 4% paraformaldehyde for 12 to 24 h and subsequently stained for BTV-8 with double immunofluorescent staining using a BTV-8 monoclonal antibody (8A3B.6, ID-Vet, Montpellier, France). All control embryos (CTRL and MEM) were negative for BTV-8 virus antigen at all time points. At 48 hpi, only 1 out of 7 infected embryos was positive for virus antigen (in all its cells). At 60 hpi, all remaining embryos (n = 6) were negative, whereas at 72 hpi and 96 hpi all embryos had 25% to 100% BTV-8-positive cells (n = 6 at 72 hpi and n = 7 at 96 hpi). Furthermore, 1 embryo at 72 hpi and 2 embryos at 96 hpi showed morphological signs of degeneration. This study has showed for the first time that hatched in vitro-produced blastocysts are susceptible for BTV-8 virus infection and replication in vitro. The relatively long time between virus infection and detection of viral antigen is in accordance with the slow replication cycle of the virus. Further research is ongoing to investigate the importance of BTV-8 infection in early embryonic death. The first author is supported by Research Foundation-Flanders.

scholarly journals The Murine Neuronal Receptor NgR1 Is Dispensable for Reovirus Pathogenesis

2021 ◽  
Author(s):  
Pavithra Aravamudhan ◽  
Camila Guzman-Cardozo ◽  
Kelly Urbanek ◽  
Olivia Welsh ◽  
Jennifer Konopka-Anstadt ◽  
...  
Keyword(s):  
Expression Patterns ◽  
Target Cells ◽  
Viral Attachment ◽  
Genetic Ablation ◽  
Murine Homolog ◽  
Intramuscular Inoculation ◽  
Neural Tissues ◽  
Mammalian Orthoreovirus ◽  
The Brain

Engagement of host receptors is essential for viruses to enter target cells and initiate infection. Expression patterns of receptors in turn dictate host and tissue tropism and disease pathogenesis during infection. Mammalian orthoreovirus (reovirus) displays serotype-dependent patterns of tropism in the murine central nervous system (CNS) that are dictated by viral attachment protein σ1. However, the receptor that mediates reovirus CNS tropism is unknown. Two proteinaceous receptors have been identified for reovirus, junctional adhesion molecule-A (JAM-A) and Nogo 66 receptor 1 (NgR1). Engagement of JAM-A is required for reovirus hematogenous dissemination but is dispensable for neural spread. To determine whether NgR1 functions in reovirus neuropathogenesis, we compared virus replication and disease following inoculation of wild-type (WT) and NgR1-/- mice. Genetic ablation of NgR1 did not alter replication of neurotropic reovirus strain T3SA- in the intestine and transmission to the brain following peroral inoculation. Viral titers in neural tissues following intramuscular inoculation, which provides access to neural dissemination routes, also were comparable in WT and NgR1-/- mice, suggesting that NgR1 is dispensable for reovirus neural spread to the CNS. The absence of both NgR1 and JAM-A also did not alter replication, neural tropism, and virulence of T3SA- following direct intracranial inoculation. In agreement with these findings, we found that the human but not the murine homolog of NgR1 functions as a receptor and confers efficient reovirus binding and infection of nonsusceptible cells in vitro. These results eliminate functions for JAM-A and NgR1 in shaping CNS tropism in mice and suggest that other receptors, yet to be identified, support this function.

scholarly journals Susceptibility of in vitro produced hatched bovine blastocysts to infection with bluetongue virus serotype 8

2011 ◽  
Vol 42 (1) ◽  
pp. 14 ◽  
Author(s):  
Leen Vandaele ◽  
Wendy Wesselingh ◽  
Kris De Clercq ◽  
Ilse De Leeuw ◽  
Herman Favoreel ◽  
...  
Keyword(s):  
Bluetongue Virus ◽  
Virus Serotype

Estrogens and progestins: molecular effects on brain cells

2010 ◽  
Vol 4 (3) ◽  
Author(s):  
Paolo Mannella ◽  
Tommaso Simoncini ◽  
Andrea Riccardo Genazzani
Keyword(s):  
Sex Steroids ◽  
Molecular Mechanisms ◽  
Neural Cells ◽  
Protective Effects ◽  
Complete Knowledge ◽  
Molecular Effects ◽  
In Vivo Effects ◽  
The Brain

AbstractSex steroids are known to regulate brain function and their role is so important that several diseases are strictly correlated with the onset of menopause when estrogen-progesterone deficiency makes neural cells much more vulnerable to toxic stimuli. Although in the past years several scientists have focused their studies on in vitro and in vivo effects of sex steroids on the brain, we are still far from complete knowledge. Indeed, contrasting results from large clinical trials have made the entire issue much more complicated. Currently we know that protective effects exerted by sex steroids depend on several factors among which the dose, the health of the cells and the type of molecule being used. In this review, we present an overview of the direct and indirect effects of estrogen and progesterone on the brain with specific focus on the molecular mechanisms by which these molecules act on neural cells.

Fish embryo culture: rhombencephalic neuritic outgrowth in explanted axial cords from the zebrafish Brachydanio rerio (Hamilton-Buchanan)

1982 ◽  
Vol 60 (12) ◽  
pp. 3215-3219 ◽  
Author(s):  
Hans W. Laale
Keyword(s):  
Embryo Culture ◽  
Fiber Bundles ◽  
Target Cells ◽  
Neural Cells ◽  
Brachydanio Rerio ◽  
Fish Embryo ◽  
Mammalian Tissues ◽  
Neuritic Outgrowth ◽  
Close Contacts

Fifteen stage 17 axial cords from the zebrafish Brachydanio rerio were stripped of epidermis and separated from their yolk spheres. Posterior halves, from the mid-rhombencephalic level to the caudal knob, were maintained in nutrient medium for 30 days at 24 ± 2 °C. The study describes preliminary observations on rhombencephalic neuritic outgrowth and associations with non-neural cells from 7 to 30 days of culture. Nerve fiber bundles increased in number and complexity with time and displayed heterogeneous branching behaviour. Frequent multiple close contacts with individual non-neural cells were observed, but selectivity of attraction to specific target cells was not verified. The findings are compared with similar contacts reported for mammalian tissues in vitro.

scholarly journals Lysozyme transport to the brain by liposomes

2018 ◽  
Vol 1 (2) ◽  
pp. 146-161 ◽  
Author(s):  
Mirjam M Nordling-David ◽  
Elior Rachamin ◽  
Etty Grad ◽  
Gershon Golomb
Keyword(s):  
Blood Brain Barrier ◽  
White Blood Cells ◽  
Brain Barrier ◽  
Immunofluorescent Staining ◽  
Phagocytic Cells ◽  
Blood Brain ◽  
The Brain ◽  
Positively Charged

Delivery of drugs into the brain is limited due to poor penetrability of many drugs via the blood-brain barrier. Previous studies have shown that the brain is kept under close surveillance by the immune system, implying that circulating phagocytic cells, such as neutrophils and monocytes, are crossing the blood-brain barrier. We hypothesized that charged liposomes could be transported to the brain following their phagocytosis by circulating monocytes. In this work, we investigated the capacity of circulating monocytes to be exploited as a drug delivery system following IV administration of nano-sized, positively fluorescently labeled liposomes containing the protein lysozyme. Negatively charged fluorescently labeled liposomes were used for comparison. By using a modified thin-film hydration technique, the desired properties of the liposomal formulations were achieved including size, polydispersity index, high drug concentration, and stability. In vitro results showed a significant time-dependent uptake of positively charged liposomes by RAW264.7 cells. In vivo results revealed that circulating white blood cells (mainly monocytes) contained high levels of fluorescently labeled liposomes. Screening of brain sections using confocal microscopy uncovered that a substantial amount of fluorescently labeled liposomes, in contrast to the fluorescent markers in solution, was transported into the brain. In addition, anti-CD68 immunofluorescent staining of brain sections demonstrated co-localization of positively charged liposomes and macrophages in different brain sections. Furthermore, significantly higher levels of lysozyme were detected in brain lysates from rats treated with positively charged liposomes compared to rats treated with lysozyme solution. Taken together this confirms our hypothesis that the designed liposomes were transported to the brain following their phagocytosis by circulating monocytes.

scholarly journals H-2-restricted cytotoxic effectors generated in vitro by the addition of trinitrophenyl-conjugated soluble proteins

1978 ◽  
Vol 147 (2) ◽  
pp. 352-368 ◽  
Author(s):  
A Schmitt-Verhulst ◽  
CB Pettinelli ◽  
PA Henkart ◽  
JK Lunney ◽  
GM Shearer
Keyword(s):  
T Cell ◽  
Spleen Cell ◽  
Cell Cultures ◽  
Sodium Dodecyl ◽  
Soluble Proteins ◽  
Immunofluorescent Staining ◽  
Cytotoxic T Cell ◽  
Target Cells ◽  
Spleen Cells

Murine spleen cells from normal donors were cultured in vitro with trinitrobenzene sulfonate (TNBS)-conjugated soluble proteins, i.e., bovine gamma globulin (TNP-BGG) or bovine serum albumin (TNP-BSA). Addition of 100 μg of any of these TNP-proteins to the spleen cell cultures led to the generation of cytotoxic T-cell effectors which were H-2-restricted and TNP- specific. The lytic potential of such effectors was comparable to that generated by sensitization with TNBS-modified syngeneic cells, and was restricted to haplotypes shared at the K or K plus I-A, or the D regions of the H-2 complex. Greater effecter cell activity was generated by addition of TNP-BGG against TNBS-modified targets which shared K plus I-A than against modified targets which shared the D region with the responding cells, which suggests that the same immune response genes are involved when the response is generated by the addition of TNP-conjugated soluble proteins or of TNBS- modified cells. H-2-restricted, TNP-specific effecter cells were generated by culturing mouse spleen cells with syngeneic cells which had been preincubated with TNP- BGG or TNP-BSA for 1.5 h. The addition of unconjugated soluble proteins to the cultures did not result in cytotoxic effectors detectable on H-2-matched targets, whether the targets were prepared by modification with TNBS, or by incubation with either the unconjugated or TNP-conjugated proteins. Depletion of phagocytic cells in the tumor preparation by Sephadex G-10 column fractionation before incubation with TNP-BSA had no effect on their lysis by the relevant effector cells. Immunofluorescent staining of tumor target cells with anti-TNP antibodies indicated that TNP could be detected on the tumor cells within 10 rain of incubation with TNP-BSA. The cytotoxic response generated by addition of the TNP-proteins to spleen cell cultures was found to be T-cell dependent at the effector phase, as shown by the sensitivity of the lytic phase to absorbed RAMB and complement. Furthermore, the response did not appear to be attributable to antibody-dependent cellular cytotoxicity. Three mechanisms were considered which could account for the generation of H-2-restricted, TNP-specific, cytotoxic T-cell effectors by the addition of soluble TNP-proteins. These include covalent linkage of activated TNP groups from the soluble proteins to cell surface components, macrophage processing of the soluble conjugates and presentation to the responding lymphocytes in association with H-2-coded self structures, or hydrophobic interaction of the TNP-proteins to cell surfaces. Results obtained from sodium dodecyl sulfate gel patterns indicating that cell-bound TNP was still linked to BSA, and the observation that phagocytic-depleted cells could interact with the soluble TNP-proteins and function as H-2-restricted targets, appear not to favor the first two proposed mechanisms.

scholarly journals Efficacy of Amphotericin B in Combination with Flucytosine against Flucytosine-Susceptible or Flucytosine-Resistant Isolates of Cryptococcus neoformans during Disseminated Murine Cryptococcosis

2006 ◽  
Vol 50 (1) ◽  
pp. 113-120 ◽  
Author(s):  
Patrick Schwarz ◽  
Françoise Dromer ◽  
Olivier Lortholary ◽  
Eric Dannaoui
Keyword(s):  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Resistant Isolate ◽  
Fungal Burden ◽  
Cryptococcal Infection ◽  
Susceptible Isolate ◽  
The Difference ◽  
The Brain ◽  
Better Than

ABSTRACT Whether or not flucytosine should be administered to patients infected with Cryptococcus neoformans isolates found to be resistant to flucytosine in vitro remains a controversial issue. Thus, the efficacy of amphotericin B and flucytosine in combination was investigated by mortality and fungal burden studies in a murine model of disseminated cryptococcosis using two clinical isolates of Cryptococcus neoformans, one susceptible and one resistant (i.e., 64 μg/ml) to flucytosine. Amphotericin B was given intraperitoneally at 0.25 or 0.5 mg/kg/day, while flucytosine was given at 100 or 250 mg/kg/day orally. Treatment was started 24 h or day 6 after inoculation and continued for 5 days in fungal burden and mortality studies, respectively. The combination of amphotericin B at 0.5 mg/kg/day and flucytosine at 250 mg/kg/day was significantly more effective than monotherapies for reducing fungal burden in brain, spleen, and lungs after infection by the flucytosine-susceptible isolate and in brain and spleen for the flucytosine-resistant isolate. For the flucytosine-resistant isolate, the combination of amphotericin B at 0.5 mg/kg/day with flucytosine at 100 mg/kg/day was significantly better than monotherapies for reducing the fungal burden in the brain. Survival obtained after the combination of amphotericin B at 0.5 mg/kg/day and flucytosine at 250 mg/kg/day increased compared to that obtained with monotherapies for both isolates, but the difference was statistically significant only for the flucytosine-susceptible isolate. Antagonism was never observed. This study demonstrates the beneficial effect of the addition of flucytosine to amphotericin B against experimental disseminated cryptococcal infection even when the C. neoformans isolate is resistant to flucytosine.

scholarly journals Autoimmune Epilepsy - Novel Multidisciplinary Analysis, Discoveries and Insights

2022 ◽  
Vol 12 ◽  
Author(s):  
Mia Levite ◽  
Hadassa Goldberg
Keyword(s):  
T Cells ◽  
Intractable Epilepsy ◽  
Cognitive Behavioral ◽  
Neural Cells ◽  
Normal Brain ◽  
Antibodies To Glutamate ◽  
Autoimmune Antibodies ◽  
Autoimmune Epilepsy ◽  
The Brain

Epilepsy affects ~50 million people. In ~30% of patients the etiology is unknown, and ~30% are unresponsive to anti-epileptic drugs. Intractable epilepsy often leads to multiple seizures daily or weekly, lasting for years, and accompanied by cognitive, behavioral, and psychiatric problems. This multidisciplinary scientific (not clinical) ‘Perspective’ article discusses Autoimmune Epilepsy from immunological, neurological and basic-science angles. The article includes summaries and novel discoveries, ideas, insights and recommendations. We summarize the characteristic features of the respective antigens, and the pathological activity in vitro and in animal models of autoimmune antibodies to: Glutamate/AMPA-GluR3, Glutamate/NMDA-NR1, Glutamate/NMDA-NR2, GAD-65, GABA-R, GLY-R, VGKC, LGI1, CASPR2, and β2 GP1, found in subpopulations of epilepsy patients. Glutamate receptor antibodies: AMPA-GluR3B peptide antibodies, seem so far as the most exclusive and pathogenic autoimmune antibodies in Autoimmune Epilepsy. They kill neural cells by three mechanisms: excitotoxicity, Reactive-Oxygen-Species, and complement-fixation, and induce and/or facilitate brain damage, seizures, and behavioral impairments. In this article we raise and discuss many more topics and new insights related to Autoimmune Epilepsy. 1. Few autoimmune antibodies tilt the balance between excitatory Glutamate and inhibitory GABA, thereby promoting neuropathology and epilepsy; 2. Many autoantigens are synaptic, and have extracellular domains. These features increase the likelihood of autoimmunity against them, and the ease with which autoimmune antibodies can reach and harm these self-proteins. 3. Several autoantigens have ‘frenetic character’- undergoing dynamic changes that can increase their antigenicity; 4. The mRNAs of the autoantigens are widely expressed in multiple organs outside the brain. If translated by default to proteins, broad spectrum detrimental autoimmunity is expected; 5. The autoimmunity can precede seizures, cause them, and be detrimental whether primary or epiphenomenon; 6. Some autoimmune antibodies induce, and associate with, cognitive, behavioral and psychiatric impairments; 7. There are evidences for epitope spreading in Autoimmune Epilepsy; 8. T cells have different ‘faces’ in the brain, and in Autoimmune Epilepsy: Normal T cells are needed for the healthy brain. Normal T cells are damaged by autoimmune antibodies to Glutamate/AMPA GluR3, which they express, and maybe by additional autoantibodies to: Dopamine-R, GABA-R, Ach-R, Serotonin-R, and Adrenergic-R, present in various neurological diseases (summarized herein), since T cells express all these Neurotransmitter receptors. However, autoimmune and/or cytotoxic T cells damage the brain; 9. The HLA molecules are important for normal brain function. The HLA haplotype can confer susceptibility or protection from Autoimmune Epilepsy; 10. There are several therapeutic strategies for Autoimmune Epilepsy.

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