Brains Of Infected Animals Research Articles

Experimental Granulomatous Amebic Encephalitis Caused by Acanthamoeba castellanii.

Acanthamoeba genus can affect humans with diseases such as granulomatous amebic encephalitis (GAE), a highly lethal neuroinfection. Several aspects of the disease still need to be elucidated. Animal models of GAE have advanced our knowledge of the disease. This work tested Wistar rats (Rattus norvegicus albinus) as an animal model of GAE. For this, 32 animals were infected with 1 × 106A. castellanii trophozoites of the T4 genotype. Ameba recovery tests were carried out using agar plates, vascular extravasation assays, behavioral tests, and histopathological technique with H/E staining. Data were subjected to linear regression analysis, one-way ANOVA, and Tukey's test, performed in the GraphPad Prism® 8.0 program, with a significance level of p < 0.05. The results revealed the efficiency of the model. Amebae were recovered from the liver, lungs, and brain of infected animals, and there were significant encephalic vascular extravasations and behavioral changes in these animals, but not in the control animals. However, not all infected animals showed positive histopathology for the analyzed organs. Nervous tissues were the least affected, demonstrating the role of the BBB in the defense of the CNS. Supported by the demonstrated evidence, we confirm the difficulties and the feasibilities of using rats as an animal model of GAE.

Continuous presence ofgenetically diverse rustrela virus lineages inyellow-necked field mouse reservoir populations innortheastern Germany.

Rustrela virus (RusV; species Rubivirus strelense, family Matonaviridae) was discovered in different zoo animal species affected by fatal encephalitis. Simultaneous RusV RNA detection in multiple yellow-necked field mice (Apodemus flavicollis) suggested this rodent as a reservoir of RusV. Here, we investigated 1,264 yellow-necked field mice and sympatric other small mammals from different regions in Germany for RusV RNA using an optimized reverse transcription-quantitative polymerase chain reaction (RT-qPCR) protocol and high-throughput sequencing. The investigation resulted in the detection of RusV RNA exclusively in 50 of 396 (12.6 per cent) yellow-necked field mice but absence in other sympatric species. RT-qPCR-determined tissue distribution of RusV RNA revealed the highest viral loads in the central nervous system, with other tissues being only very rarely affected. The histopathological evaluation did not reveal any hints of encephalitis in the brains of infected animals despite the detection of viral RNA in neurons by in situ hybridization (ISH). The positive association between the body mass of yellow-necked field mice and RusV RNA detection suggests a persistent infection. Phylogenetic analysis of partial E1 and full-genome sequences showed a high diversification with at least four RusV lineages (1A-1D) in northeastern Germany. Moreover, phylogenetic and isolation-by-distance analyses indicated evolutionary processes of RusV mostly in local reservoir populations. A comparison of complete genome sequences from all detected RusV lineages demonstrated a high level of amino acid and nucleotide sequence variability within a part of the p150 peptide of the non-structural polyprotein and its coding sequence, respectively. The location of this region within the RusV genome and its genetic properties were comparable to the hypervariable region of the rubella virus. The broad range of detected RusV spillover hosts in combination with its geographical distribution in northeastern Germany requires the assessment of its zoonotic potential and further analysis of encephalitis cases in mammals. Future studies have to prove a putative co-evolution scenario for RusV in the yellow-necked field mouse reservoir.

Host Desmin Interacts with RABV Matrix Protein and Facilitates Virus Propagation.

Microfilaments and microtubules, two crucial structures of cytoskeletal networks, are usurped by various viruses for their entry, egress, and/or intracellular trafficking, including the Rabies virus (RABV). Intermediate filaments (IFs) are the third major component of cytoskeletal filaments; however, little is known about the role of IFs during the RABV infection. Here, we identified the IF protein desmin as a novel host interactor with the RABV matrix protein, and we show that this physical interaction has a functional impact on the virus lifecycle. We found that the overexpression of desmin facilitates the RABV infection by increasing the progeny virus yield, and the suppression of endogenous desmin inhibits virus replication. Furthermore, we used confocal microscopy to observe that the RABV-M co-localizes with desmin in IF bundles in the BHK-21 cells. Lastly, we found that mice challenged with RABV displayed an enhanced expression of desmin in the brains of infected animals. These findings reveal a desmin/RABV-M interaction that positively regulates the virus infection and suggests that the RABV may utilize cellular IFs as tracks for the intracellular transport of viral components and efficient budding.

Limited cross-variant immunity from SARS-CoV-2 Omicron without vaccination

SARS-CoV-2 Delta and Omicron are globally relevant variants of concern. Although individuals infected with Delta are at risk of developing severe lung disease, infection with Omicron often causes milder symptoms, especially in vaccinated individuals1,2. The question arises of whether widespread Omicron infections could lead to future cross-variant protection, accelerating the end of the pandemic. Here we show that without vaccination, infection with Omicron induces a limited humoral immune response in mice and humans. Sera from mice overexpressing the human ACE2 receptor and infected with Omicron neutralize only Omicron, but not other variants of concern, whereas broader cross-variant neutralization was observed after WA1 and Delta infections. Unlike WA1 and Delta, Omicron replicates to low levels in the lungs and brains of infected animals, leading to mild disease with reduced expression of pro-inflammatory cytokines and diminished activation of lung-resident T cells. Sera from individuals who were unvaccinated and infected with Omicron show the same limited neutralization of only Omicron itself. By contrast, Omicron breakthrough infections induce overall higher neutralization titres against all variants of concern. Our results demonstrate that Omicron infection enhances pre-existing immunity elicited by vaccines but, on its own, may not confer broad protection against non-Omicron variants in unvaccinated individuals.

Dual Anti-Malarial and GSK3β-Mediated Cytokine-Modulating Activities of Quercetin Are Requisite of Its Potential as a Plant-Derived Therapeutic in Malaria

Although death in malaria is attributed to cerebrovascular blockage and anaemia, overwhelming cytokine production can contribute to the severity of the disease. Therefore, mitigation of dysregulated inflammatory signalling may provide further benefit for malaria treatment. Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is known to inhibit glycogen synthase kinase-3β (GSK3β), a potent regulator of both pro- and anti-inflammatory effects. Quercetin is therefore a potential therapeutic to modulate the imbalanced cytokine production during malarial infection. Anti-malarial effects of quercetin were evaluated in murine models of severe and cerebral malaria using Plasmodium berghei NK65 and ANKA strains, respectively. Western blotting and analysis of cytokines were carried out to determine the GSK3β-mediated cytokine-modulating effects of quercetin in infected animals. Quercetin (25 mg/kg BW) treatment in P. berghei NK65-infected animals resulted in 60.7 ± 2.4% suppression of parasitaemia and significantly decreased serum levels of TNF-α and IFN-γ, whilst levels of IL-10 and IL-4 were elevated significantly. Western analysis revealed that pGSK3β (Ser9) increased 2.7-fold in the liver of quercetin-treated NK65-infected animals. Treatment of P. berghei ANKA-infected mice with quercetin (15 mg/kg BW) increased (2.3-fold) pGSK3β (Ser9) in the brains of infected animals. Quercetin is a potential plant-derived therapeutic for malaria on the basis that it can elicit anti-malarial and GSK3β-mediated cytokine-modulating effects.

Necroptosis and Caspase-2-Mediated Apoptosis of Astrocytes and Neurons, but Not Microglia, of Rat Hippocampus and Parenchyma Caused by Angiostrongylus cantonensis Infection.

Infection with the roundworm Angiostrongylus cantonensis is the main cause of eosinophilic meningitis worldwide. The underlying molecular basis of the various pathological outcomes in permissive and non-permissive hosts infected with A. cantonensis remains poorly defined. In the present study, the histology of neurological disorders in the central nervous system (CNS) of infected rats was assessed by using hematoxylin and eosin staining. Quantitative reverse transcription polymerase chain reaction (RT-qPCR), western blot and immunofluorescence (IF) were used in evolutions of the transcription and translation levels of the apoptosis-, necroptosis-, autophagy-, and pyroptosis-related genes. The distribution of apoptotic and necroptotic cells in the rat hippocampus and parenchyma was further detected using flow cytometry, and the features of the ultrastructure of the cells were examined by transmission electron microscopy (TEM). The inflammatory response upon CNS infection with A. cantonensis evolved, as characterized by the accumulation of a small number of inflammatory cells under the thickened meninges, which peaked at 21 days post-infection (dpi) and returned to normal by 35 dpi. The transcription levels and translation of caspase-2, caspase-8, RIP1 and RIP3 increased significantly at 21 and 28 dpi but decreased sharply at 35 dpi compared to those in the normal control group. However, the changes in the expression of caspase-1, caspase-3, caspase-11, Beclin-1 and LC3B were not obvious, suggesting that apoptosis and necroptosis but not autophagy or pyroptosis occurred in the brains of infected animals at 21 and 28 dpi. The results of RT-qPCR, western blot analysis, IF, flow cytometry and TEM further illustrated that necroptosis and caspase-2-mediated apoptosis occurred in astrocytes and neurons but not in microglia in the parenchyma and hippocampus of infected animals. This study provides the first evidence that neuronal and astrocytic necroptosis and caspase-2-mediated apoptosis are induced by A. cantonensis infection in the parenchymal and hippocampal regions of rats at 21 and 28 dpi but these processes are negligible at 35 dpi. These findings enhance our understanding of the pathogenesis of A. cantonensis infection and provide new insights into therapeutic approaches targeting the occurrence of cell death in astrocytes and neurons in infected patients.

Experimental infection by Neospora caninum in gerbil reduces activity of enzymes involved in energy metabolism

Neospora caninum is a protozoan that has tropism for the central nervous system. The aim of this study was to determine whether experimental infection of gerbils would interfere with activity of enzymes associated with energy metabolism. We randomized 20 gerbils into two groups (ten animals per group): the control group (healthy animals; uninfected) and the infected group (experimentally infected with dose 7.8 × 102 tachyzoites of N. caninum per gerbil). On day six and twelve post-infection (PI), brain and spleen tissues were collected for biochemical and histopathological analyses. No histopathological lesions were observed in the brains of infected animals; however, inflammatory infiltrates were found in the spleen. Significantly greater levels of reactive oxygen species (ROS) were observed in the brain and spleen of infected gerbils than in the control group at 12 days PI. Cytosolic creatine kinase (CK-CYT), mitochondrial creatine kinase (CK-MIT), and pyruvate kinase (PK) activities were lower in the brains of infected gerbils than in those of the control group on day 12 PI. There was significantly less CK-CYT activity in the spleens of infected gerbils on day 6 and 12 PI. Finally, there was significantly less sodium-potassium ion pump (Na+/K+ ATPase) activity in the brains and spleens of infected gerbils on day 12 PI. These data suggest that experimental infection with N. caninum interfered with energy metabolism associated with ATP homeostasis in the brain and spleen, directly or indirectly, apparently mediated by ROS overproduction, contributing to inhibition of Na+/K+ ATPase activity.

Experimental Infection by Brucella ovis: Changes in NTPDase, 5'-Nucleotidase and Acetylcholinesterase Associated Cerebral Oxidative Stres

Background: Changes in purinergic and cholinergic signaling have been demonstrated in various pathologies associated with inflammation; however, the changes in brucellosis caused by the Gram-negative coccobacillus Brucella ovis are not known. B. ovis is generally asymptomatic in sheep. Hepatosplenomegaly has been described in B. ovis, a non-zoonotic species, characterized by an extravascular inflammatory response. Purinergic system enzymes are closely involved with the modulation of the immune system, pro- and anti-inflammatory events. The objective of this study was to investigate the role of ectonucleotidases and cholinesterase’s in the brains of mice experimentally infected with B. ovis.Materials, Methods &amp; Results: Forty-eight animals were divided into two groups: control (n = 24) and infected (n = 24). In group infected, 100 µL containing 1.3 x 107 UFC B. ovis /mL via intraperitoneal was used in inoculation. The brains were collected from the animals on days 7, 15, 30 and 60 post-infection (PI). We measured levels of TBARS (substances reactive to thiobarbituric acid) and ROS (reactive oxygen species) in the brain. The activity of NTPDase (using ATP and ADP as substrate) and 5'-nucleotidase (using AMP as substrate) were evaluated in brain in addition to histopathological analysis. No histopathological lesions were observed in the control group nor the infected group at days 7, 15, and 30 PI. However,multifocal areas with moderate microgliosis and inflammatory infiltrates in the cerebral cortex were observed at day 60 PI in the infected animals. B. ovis DNA was detected in brain. During the course of infection, B. ovis caused greater lipid peroxidation in the brains of infected animals than in the control group at day 60PI. No significant results were observed at 7, 15 or day 30 PI. Similarly, there was significantly more reactive oxygen species at day 60 PI in brains of infected animals than in the control group. NTPDase activity (using ATP and AMP as substrate) was lower at days 7 and 15 PI in infected animals than in control. However, during the course of infection there was an increase in NTPDase activity at day 60 PI in the infected group. The infected animals showed a decrease of 5´-nucleotidase (AMP as substrate) activity at days 7 and 30 PI. On the other hand, 5´-nucleotidase activity was greater on day 60 PI in the experimental group than in the control. The results suggest that nucleotide hydrolysis was low in the acute phase (up to day 30 PI) due to the decrease of NTPDase and 5´-nucleotidase activities. After day 60 PI, there was a reversal in enzyme activities, probably with concomitant increase of extracellular nucleotides. AChE activity in brain on days 30 and 60 PI compared to control.Discussion: Among the functions of NTPDase are inhibition of platelet aggregation, vascular homeostasis, modulation of inflammation and immune response, all via its regulation of extracellular concentrations of ATP, a pro-inflammatory molecule. E-NTPDase plays an important role in controlling lymphocyte function, including antigen recognition and activation of cytotoxic T cell effector functions, as well as the generation of signals. The enzyme E-5´-nucleotidase also exerts non-enzymatic functions, including induction of intracellular signaling and mediation of cell-cell adhesion and cell-matrix and migration. Levels of acetylcholine are regulated by cholinesterase enzymes that are present in cholinergic and noncholinergic tissues, as the acetylcholinesterase (AChE) is a membrane-bound enzyme, primarily found in the brain and cholinergic neurons, where it participates in the structural regulation of postsynaptic differentiation. The results demonstrated that the chronicity of infection by B. ovis causes oxidative damage and inflammation in the brain, as well as modulation of ectonucleotidases and AChE activities.

Pathogenicity, genotyping and antibacterial susceptibility of the Listeria spp. recovered from stray dogs

The present study aimed to determine the prevalence of Listeria spp. in stray dogs in the Kayseri province of Turkey. In addition, serotyping, genotyping and virulence gene analysis of the isolated Listeria spp. were performed and their pathogenicity and antibacterial susceptibility were investigated.The study included 80 rectal swaps taken from 80 stray dogs of different ages and gender that were sheltered in the Kayseri Municipal Dog Shelter. Listeria selective broth and Listeria selective agar were used for the isolation of Listeria spp. and the isolates were identified using a Microbact 12L (Oxoid, England) identification test kit. 16S rDNA sequencing and species-specific polymerase chain reaction (PCR) were performed for molecular identification of the isolates, multiplex PCR and a serological test were performed for serotyping, and PCR was used for virulence gene analysis. For determining the pathogenicity of L. monocytogenes and L. innocua isolates, a total of 100 mice (50 pregnant and 50 non-pregnant) were used. The mice were infected intraperitoneally; the inoculation dose was 1 × 109 CFU/mL and 0.2 mL was used for each animal. Tissue samples obtained from infected mice were processed for the re-isolation of the Listeria spp. and then stained with hematoxylin eosin and Brown-Brenn Gram stain. The antibiotic susceptibilities of the isolates were determined by the disk diffusion method.Listeria spp. were isolated from 5 (6.25%) of the 80 fecal samples. While 1 of the isolates was identified as L. monocytogenes, 4 of them were identified as L. innocua. Serotyping by serological and molecular methods revealed the isolate of L. monocytogenes to be serotype 1/2a. According to the phylogenetic trees, L. innocua and L. monocytogenes strains were clustered in different groups. The L. monocytogenes isolate was positive for all virulence genes tested. All L. innocua isolates were positive for the plcB gene. While all L. innocua isolates were negative for the lin1068 gene, 3 L. innocua isolates were found to be positive for the lin0558 gene. In mice infected with L. monocytogenes, pathological findings were observed in the uterus, intestines, pancreas, and heart. In mice infected with L. innocua, pathological findings were observed in the stomach, intestines and spleen. L. monocytogenes- or L. innocua-related infections or other inflammatory reactions were not observed in the brains of infected animals. On histopathological examination with Gram stain, an abundance of Listeria spp. was observed in the lesions of the liver, spleen, uterus, and kidney. Moreover, while abortion was observed in all animals infected with L. monocytogenes, it was not observed in any of the animals infected with L. innocua. Antibiotic susceptibility testing revealed that all 5 isolates were sensitive to ampicillin, amoxicillin/clavulanic acid, erythromycin, gentamicin, penicillin G, and trimethoprim-sulfamethoxazole and were resistant to nalidixic acid, streptomycin, and cefuroxime sodium.Considering also the pathogenicity of the isolated microorganisms, it can be suggested that stray dogs as carriers of Listeria spp. are a significant risk to public health. As L. innocua isolates, which are considered apathogenic, led to the occurrence of lesions similar to those caused by L. monocytogenes, detailed studies on the pathogenesis of L. innocua infections caused by L. innocua isolates recovered from various sources are required.

Serum and brain purine levels in an experimental systemic infection of mice by Cryptococcus neoformans: Purinergic immunomodulatory effects

The aim of this study was to evaluate the purine levels in serum and brains of mice experimentally infected by Cryptococcus neoformans. Twenty-four mice were divided into the following groups: a control group (n = 12; Group A) and an infection group with animals that were infected (n = 12; Group B) with a 0.3-mL intraperitoneal injection containing 1.7 × 107C. neoformans cells. Blood and brains were collected on days 20 (n = 6 per group) and 50 (n = 6 per group) post-infection (PI). Histopathology and lung and brain cultures were performed to confirm fungal infection and tissue injuries. The levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine (ADO), inosine (INO), hypoxanthine (HYPO), xanthine (XAN) and uric acid (UA) in brains and serum were measured by high-performance liquid chromatography (HPLC) analysis. At both time points, histopathology analysis revealed inflammatory infiltrates in the brains and lungs of infected mice; clinical signs, such as piloerection and clinical respiratory distress, were also observed. ATP levels were significantly increased on days 20 and 50 PI (P < 0.01) in brains and serum, while brain ADO levels were increased on day 20 PI; brain and serum ADO levels were decreased on day 50 PI. Levels of ADP and AMP did not differ between groups (P > 0.05). Serum levels of INO of infected mice increased only on day 50 PI (P < 0.05). HYPO levels were reduced in the brains of infected animals at both experimental time points and were decreased in serum at day 50 PI (P < 0.05). XAN levels increased in infected mice only in serum on day 50 PI (P < 0.05). The endogenous anti-oxidant uric acid was significantly increased in brain (days 20 and 50 PI) and decreased in serum. It is possible that C. neoformans infection in mice leads to a high ATP/ADO ratio that may improve the brain pro-inflammatory response during both periods, while high ATP levels in serum act as a systemic signal to improve the immune response. Moreover, the anti-oxidant uric acid may increase in the brain to protect inflamed tissue from oxidative stress.

Exploring virulence and immunogenicity in the emerging pathogen Sporothrix brasiliensis.

Sporotrichosis is a polymorphic chronic infection of humans and animals classically acquired after traumatic inoculation with soil and plant material contaminated with Sporothrix spp. propagules. An alternative and successful route of transmission is bites and scratches from diseased cats, through which Sporothrix yeasts are inoculated into mammalian tissue. The development of a murine model of subcutaneous sporotrichosis mimicking the alternative route of transmission is essential to understanding disease pathogenesis and the development of novel therapeutic strategies. To explore the impact of horizontal transmission in animals (e.g., cat-cat) and zoonotic transmission on Sporothrix fitness, the left hind footpads of BALB/c mice were inoculated with 5×106 yeasts (n = 11 S. brasiliensis, n = 2 S. schenckii, or n = 1 S. globosa). Twenty days post-infection, our model reproduced both the pathophysiology and symptomology of sporotrichosis with suppurating subcutaneous nodules that progressed proximally along lymphatic channels. Across the main pathogenic members of the S. schenckii clade, S. brasiliensis was usually more virulent than S. schenckii and S. globosa. However, the virulence in S. brasiliensis was strain-dependent, and we demonstrated that highly virulent isolates disseminate from the left hind footpad to the liver, spleen, kidneys, lungs, heart, and brain of infected animals, inducing significant and chronic weight loss (losing up to 15% of their body weight). The weight loss correlated with host death between 2 and 16 weeks post-infection. Histopathological features included necrosis, suppurative inflammation, and polymorphonuclear and mononuclear inflammatory infiltrates. Immunoblot using specific antisera and homologous exoantigen investigated the humoral response. Antigenic profiles were isolate-specific, supporting the hypothesis that different Sporothrix species can elicit a heterogeneous humoral response over time, but cross reaction was observed between S. brasiliensis and S. schenckii proteomes. Despite great diversity in the immunoblot profiles, antibodies were mainly derived against 3-carboxymuconate cyclase, a glycoprotein oscillating between 60 and 70 kDa (gp60-gp70) and a 100-kDa molecule in nearly 100% of the assays. Thus, our data broaden the current view of virulence and immunogenicity in the Sporothrix-sporotrichosis system, substantially expanding the possibilities for comparative genomic with isolates bearing divergent virulence traits and helping uncover the molecular mechanisms and evolutionary pressures underpinning the emergence of Sporothrix virulence.

Free Sialic Acid Acts as a Signal That Promotes Streptococcus pneumoniae Invasion of Nasal Tissue and Nonhematogenous Invasion of the Central Nervous System.

Streptococcus pneumoniae (pneumococcus) is a leading cause of bacterial meningitis and neurological sequelae in children worldwide. Acute bacterial meningitis is widely considered to result from bacteremia that leads to blood-brain barrier breakdown and bacterial dissemination throughout the central nervous system (CNS). Previously, we showed that pneumococci can gain access to the CNS through a nonhematogenous route without peripheral blood infection. This access is thought to occur when the pneumococci in the upper sinus follow the olfactory nerves and enter the CNS through the olfactory bulbs. In this study, we determined whether the addition of exogenous sialic acid postcolonization promotes nonhematogenous invasion of the CNS. Previously, others showed that treatment with exogenous sialic acid post-pneumococcal infection increased the numbers of CFU recovered from an intranasal mouse model of infection. Using a pneumococcal colonization model, an in vivo imaging system, and a multiplex assay for cytokine expression, we demonstrated that sialic acid can increase the number of pneumococci recovered from the olfactory bulbs and brains of infected animals. We also show that pneumococci primarily localize to the olfactory bulb, leading to increased expression levels of proinflammatory cytokines and chemokines. These findings provide evidence that sialic acid can enhance the ability of pneumococci to disseminate into the CNS and provide details about the environment needed to establish nonhematogenous pneumococcal meningitis.

Glial cell activation, recruitment, and survival of B-lineage cells following MCMV brain infection

BackgroundChemokines produced by reactive glia drive migration of immune cells and previous studies from our laboratory have demonstrated that CD19+ B cells infiltrate the brain. In this study, in vivo and in vitro experiments investigated the role of reactive glial cells in recruitment and survival of B-lineage cells in response to (murine cytomegalovirus) MCMV infection.MethodsFlow cytometric analysis was used to assess chemokine receptor expression on brain-infiltrating B cells. Real-time RT-PCR and ELISA were used to measure chemokine levels. Dual-immunohistochemical staining was used to co-localize chemokine production by reactive glia. Primary glial cell cultures and migration assays were used to examine chemokine-mediated recruitment. Astrocyte: B cell co-cultures were used to investigate survival and proliferation.ResultsThe chemokine receptors CXCR3, CXCR5, CCR5, and CCR7 were detected on CD19+ cells isolated from the brain during MCMV infection. In particular, CXCR3 was found to be elevated on an increasing number of cells over the time course of infection, and it was the primary chemokine receptor expressed at 60 days post infection Quite different expression kinetics were observed for CXCR5, CCR5, and CCR7, which were elevated on the highest number of cells early during infection and decreased by 14, 30, and 60 days post infection Correspondingly, elevated levels of CXCL9, CXCL10, and CXCL13, as well as CCL5, were found within the brains of infected animals, and only low levels of CCL3 and CCL19 were detected. Differential expression of CXCL9/CXCL10 and CXCL13 between microglia and astrocytes was apparent, and B cells moved towards supernatants from MCMV-infected microglia, but not astrocytes. Pretreatment with neutralizing Abs to CXCL9 and CXCL10 inhibited this migration. In contrast, neutralizing Abs to the ligand of CXCR5 (i.e., CXCL13) did not significantly block chemotaxis. Proliferation of brain-infiltrating B cells was detected at 7 days post infection and persisted through the latest time tested (60 days post infection). Finally, astrocytes produce BAFF (B cell activating factor of the TNF family) and promote proliferation of B cells via cell-to-cell contact.ConclusionsCXCR3 is the primary chemokine receptor on CD19+ B cells persisting within the brain, and migration to microglial cell supernatants is mediated through this receptor. Correspondingly, microglial cells produce CXCL9 and CXCL10, but not CXCL13. Reactive astrocytes promote B cell proliferation.

From Scientific Curiosity to Public Enemy Number One in Six Short Months.

From Scientific Curiosity to Public Enemy Number One in Six Short Months.

Brain Invasion by CD4(+) T Cells Infected with a Transmitted/Founder HIV-1BJZS7 During Acute Stage in Humanized Mice.

Human immunodeficiency virus (HIV)-associated neurocognitive disorder (HAND) is one of the common causes of cognitive dysfunction and morbidity among infected patients. However, to date, it remains unknown if a transmitted/founder (T/F) HIV-1 leads to neurological disorders during acute phase of infection. Since it is impossible to answer this question in humans, we studied NOD.Cg-Prkdc scid Il2rgtm1Wjl/SzJ mice (NSG) reconstituted with human PBMC (NSG-HuPBL), followed by the peritoneal challenge with the chronic HIV-1JR-FL and the T/F HIV-1BJZS7, respectively. By measuring viral load, P24 antigenemia and P24(+) cells in peripheral blood and various tissue compartments, we found that systemic infections were rapidly established in NSG-HuPBL mice by both HIV-1 strains. Although comparable peripheral viral loads were detected during acute infection, the T/F virus appeared to cause less CD4(+) T cell loss and less numbers of infected cells in different organs and tissue compartments. Both viruses, however, invaded brains with P24(+)/CD3(+) T cells detected primarily in meninges, cerebral cortex and perivascular areas. Critically, brain infections with HIV-1JR-FL but not with HIV-1BJZS7 resulted in damaged neurons together with activated microgliosis and astrocytosis as determined by significantly increased numbers of Iba1(+) microglial cells and GFAP(+) astrocytes, respectively. The increased Iba1(+) microglia was correlated positively with levels of P24 antigenemia and negatively with numbers of NeuN(+) neurons in brains of infected animals. Our findings, therefore, indicate the establishment of two useful NSG-HuPBL models, which may facilitate future investigation of mechanisms underlying HIV-1-induced microgliosis and astrocytosis.

P60 – 2600: Apoptosis in murine herpesvirus 68 encephalitis

Objective Apoptosis is assumed to contribute not only to neuronal damage but also to brain protection (destruction of infected cells) in cerebral viral infection. This study focused on anti- and proapoptotic mechanisms in the murine herpesvirus 68 (MHV-68) encephalitis/mouse model. Methods BALB/c mice inoculated neonatally with MHV-68 were sacrificed between 5 and 94 days of age. Viral (antiapoptotic M11 gene) and cellular (proapoptotic: Bax, Caspase 3, 8 and 9; antiapoptotic: Bcl-2) mechanisms were studied in brain tissue by means of PCR and morphological studies (TUNEL) and compared with findings in lung and liver tissues. Results Compared to controls, three weeks after inoculation with MHV-68 brains of infected animals show significantly increased Bcl-2 as well as Caspase 3-, 8- and 9- expression. No difference was found for BAX expression. Expression of the antiapoptotic viral gene M11 was present in 66% of the brains of infected animals. Lung and liver tissues did not show a similarly strong upregulation of different pro- and antiapoptotic proteins. No strong correlation between viral loads and the expression of different pro- and antiapoptotic genes was found. TUNEL investigations confirmed the presence of apoptosis in brains of infected mice. Conclusion Virus-related and host-related pro- and antiapoptotic mechanisms are characteristic of MHV-68 infection in mice. Upregulation of different caspases is stronger in brain than in lung and liver tissues, suggesting a specific impact of apoptosis on cerebral viral infection. The clinical impact of distinct proand antiapoptotic mechanisms is still unknown.

Immunopathological changes in the brain of immunosuppressed mice experimentally infected with Toxocara canis.

Toxocariasis is a soil-transmitted helminthozoonosis due to infection of humans by larvae of Toxocara canis. The disease could produce cognitive and behavioral disturbances especially in children. Meanwhile, in our modern era, the incidence of immunosuppression has been progressively increasing due to increased incidence of malignancy as well as increased use of immunosuppressive agents. The present study aimed at comparing some of the pathological and immunological alterations in the brain of normal and immunosuppressed mice experimentally infected with T. canis. Therefore, 180 Swiss albino mice were divided into 4 groups including normal (control) group, immunocompetent T. canis-infected group, immunosuppressed group (control), and immunosuppressed infected group. Infected mice were subjected to larval counts in the brain, and the brains from all mice were assessed for histopathological changes, astrogliosis, and IL-5 mRNA expression levels in brain tissues. The results showed that under immunosuppression, there were significant increase in brain larval counts, significant enhancement of reactive gliosis, and significant reduction in IL-5 mRNA expression. All these changes were maximal in the chronic stage of infection. In conclusion, the immunopathological alterations in the brains of infected animals were progressive over time, and were exaggerated under the effect of immunosuppression as did the intensity of cerebral infection.

Testing effects of glatiramer acetate and fingolimod in an infectious model of CNS immune surveillance

Immune surveillance of the CNS is critical for preventing infections; however, there is no accepted experimental model to assess the risk of infection when utilizing disease-modifying agents. We tested two approved agents for patients with multiple sclerosis (MS), glatiramer acetate and fingolimod, in an experimental model of CNS immune surveillance. C57BL/6 mice were infected with the ME49 strain of the neuroinvasive parasite Toxoplasma gondii (T. gondii) and then treated with GA and fingolimod. Neither treatment affected host survival; however, differences were observed in parasite load and in leukocyte numbers in the brains of infected animals. Here we demonstrate that this model could be a useful tool for analyzing immune surveillance.

Human Herpesvirus 6A Infection in CD46 Transgenic Mice: Viral Persistence in the Brain and Increased Production of Proinflammatory Chemokines via Toll-Like Receptor 9

Human herpesvirus 6 (HHV-6) is widely spread in the human population and has been associated with several neuroinflammatory diseases, including multiple sclerosis. To develop a small-animal model of HHV-6 infection, we analyzed the susceptibility of several lines of transgenic mice expressing human CD46, identified as a receptor for HHV-6. We showed that HHV-6A (GS) infection results in the expression of viral transcripts in primary brain glial cultures from CD46-expressing mice, while HHV-6B (Z29) infection was inefficient. HHV-6A DNA persisted for up to 9 months in the brain of CD46-expressing mice but not in the nontransgenic littermates, whereas HHV-6B DNA levels decreased rapidly after infection in all mice. Persistence in the brain was observed with infectious but not heat-inactivated HHV-6A. Immunohistological studies revealed the presence of infiltrating lymphocytes in periventricular areas of the brain of HHV-6A-infected mice. Furthermore, HHV-6A stimulated the production of a panel of proinflammatory chemokines in primary brain glial cultures, including CCL2, CCL5, and CXCL10, and induced the expression of CCL5 in the brains of HHV-6A-infected mice. HHV-6A-induced production of chemokines in the primary glial cultures was dependent on the stimulation of toll-like receptor 9 (TLR9). Finally, HHV-6A induced signaling through human TLR9 as well, extending observations from the murine model to human infection. Altogether, this study presents a first murine model for HHV-6A-induced brain infection and suggests a role for TLR9 in the HHV-6A-initiated production of proinflammatory chemokines in the brain, opening novel perspectives for the study of virus-associated neuropathology. HHV-6 infection has been related to neuroinflammatory diseases; however, the lack of a suitable small-animal infection model has considerably hampered further studies of HHV-6-induced neuropathogenesis. In this study, we have characterized a new model for HHV-6 infection in mice expressing the human CD46 protein. Infection of CD46 transgenic mice with HHV-6A resulted in long-term persistence of viral DNA in the brains of infected animals and was followed by lymphocyte infiltration and upregulation of the CCL5 chemokine in the absence of clinical signs of disease. The secretion of a panel of chemokines was increased after infection in primary murine brain glial cultures, and the HHV-6-induced chemokine expression was inhibited when TLR9 signaling was blocked. These results describe the first murine model for HHV-6A-induced brain infection and suggest the importance of the TLR9 pathway in HHV-6A-initiated neuroinflammation.

Eosinophil chemotactic chemokine profilings of the brain from permissive and non-permissive hosts infected with Angiostrongylus cantonenis

Angiostrongylus cantonensis invasion primarily cause heavy or negligible eosinophic meningitis and meningoencephalitis in the brain of non-permissive and permissive hosts, respectively. Chemokines are effective leukocyte chemoattractants and may play an essential role in mediating eosinophil recruitment in angiostrongyliasis. In the present study, we comparatively analyzed changes in peripheral and CSF eosinophil counts, and expression profilings of eosinophil chemotactic chemokines in A. cantonensis-infected mice (CCL 2, CCL 3, CCL 5, CCL7, CCL 8, CCL 11, CCL 12, CCL 24 and CCL 28) and rats (CCL 2, CCL 3, CCL 5, CCL 11 and CCL 12) were explored at 1, 2, 5, 7, 14, and 21 days post-infection (dpi), and found significantly elevated numbers of eosinophils in blood and CSF of infected mice after 5 dpi, while significant increases of eosinophils in blood and CSF of infected rats were detected after 5 and 14 dpi, respectively. The kinetics of CSF eosinophilia is basically correlated with eosinophil chemotactic chemokine levels in brains of infected animals at each time point. Interestingly, less CSF eosinophils and infiltration of eosinophils in the brain were noted in rats than in mice, though extremely high levels of chemokines were also maintained in the brains of infected rats at 21 dpi. We further described CCL 11 (eotaxin), a previously reported eosinophil chemotactic factor in angiostrongyliasis, was mainly released from activated microglia in mice and rats infected with A. cantonensis. Our results reveal that different complicated chemokine networks mediate recruitment of eosinophils between permissive and non-permissive hosts during A. cantonensis infection, and provide promising targets for clinical treatment of angiostrongyliasis.