Receptor for advanced glycation end products up-regulation in cerebral endothelial cells mediates cerebrovascular-related amyloid ß accumulation after Porphyromonas gingivalis infection.

Cerebrovascular-related amyloidogenesis is found in over 80% of Alzheimer's disease (AD) cases, and amyloid ß (Aß) generation is increased in the peripheral macrophages during infection of Porphyromonas gingivalis (P. gingivalis), a causal bacterium for periodontitis. In this study, we focused on receptor for advanced glycation end products (RAGE), the key molecule involves in Aß influx after P. gingivalis infection to test our hypothesis that Aß transportation from periphery into the brain, known as "Aß influx," is enhanced by P. gingivalis infection. Using cultured hCMEC/D3 cell line, in comparison to uninfected cells, directly infection with P. gingivalis (multiplicity of infection, MOI = 5) significantly increased a time-dependent RAGE expression resulting in a dramatic increase in Aß influx in the hCMEC/D3 cells; the P. gingivalis-up-regulated RAGE expression was significantly decreased by NF-κB and Cathepsin B (CatB)-specific inhibitors, and the P.gingivalis-increased IκBα degradation was significantly decreased by CatB-specific inhibitor. Furthermore, the P. gingivalis-increased Aß influx was significantly reduced by RAGE-specific inhibitor. Using 15-month-old mice (C57BL/6JJmsSlc, female), in comparison to non-infection mice, systemic P. gingivalis infection for three consecutive weeks (1 × 108  CFU/mouse, every 3 days, intraperitoneally) significantly increased the RAGE expression in the CD31-positive endothelial cells and the Aß loads around the CD31-positive cells in the mice's brains. The RAGE expression in the CD31-positive cells was positively correlated with the Aß loads. These observations demonstrate that the up-regulated RAGE expression in cerebral endothelial cells mediates the Aß influx after P. gingivalis infection, and CatB plays a critical role in regulating the NF-κB/RAGE expression. Cover Image for this issue: https://doi.org/10.1111/jnc.15073.

Effects of ketamine on the fetal transcriptomic response to umbilical cord occlusion: comparison with hypoxic hypoxia in the cerebral cortex.

KEY POINTS: The cerebral response to fetal asphyxia is characterized by an upregulation of nucleic acid and chromatin modification processes, as well as a downregulation of metabolic processes at 1 h post-umbilical cord occlusion (UCO). Twenty-four hours post UCO, there was an upregulation of metabolic processes and protein modifications. UCO did not alter bacterial gene expression levels, nor did it produce a robust inflammatory response compared to maternal hypoxia. The administration of ketamine produced minimal effects on the fetal response to UCO in the cerebral cortex. ABSTRACT: Umbilical cord occlusion (UCO) is known to cause neurological disorders in the neonate. Previously, we have reported that hypoxic hypoxia (HH) stimulates the appearance of bacteria in the fetal brain and upregulates the expression of inflammatory markers in fetal cerebral cortex (CTX) and also that ketamine attenuates these responses. In the present study, we aimed to test the hypothesis that UCO, similar to HH, produces an inflammatory response in the fetal CTX and also that treatment with ketamine reduces these effects. In chronically instrumented fetal sheep (∼125 days), 30 min of partial UCO decreased fetal PaO2 levels by ∼50%. Half of the fetuses received ketamine (3 mg kg-1 ) 10 min prior to UCO (n = 4 per group). Fetal brains were collected 1 and 24 h after the experiment and mRNA was extracted and hybridized for microarray analyses. Differentially-expressed genes were analysed for significant association with gene ontologies and pathways. After 1 h, UCO upregulated nucleic acid processing and chromatin modification and downregulated metabolic processes compared to control. After 24 h, UCO upregulated metabolic and protein modification processes. Ketamine produced minimal effects. UCO did not alter the abundance of bacterial DNA in fetal brain, nor did it upregulate inflammation pathways compared to HH. We conclude that UCO produced time-dependent responses that did not include bacterial invasion or upregulation of inflammation pathways in fetal CTX. This contrasts with the response to HH, which resulted in the appearance of bacteria in the CTX and upregulated inflammation pathways. These responses in fetal CTX to oxygen deprivation are therefore modified by the maternal or placental response to the stimulus.

An acidic microenvironment increases NK cell killing of Cryptococcus neoformans and Cryptococcus gattii by enhancing perforin degranulation.

Cryptococcus gattii and Cryptococcus neoformans are encapsulated yeasts that can produce a solid tumor-like mass or cryptococcoma. Analogous to malignant tumors, the microenvironment deep within a cryptococcoma is acidic, which presents unique challenges to host defense. Analogous to malignant cells, NK cells kill Cryptococcus. Thus, as in tumor defense, NK cells must kill yeast cells across a gradient from physiologic pH to less than 6 in the center of the cryptococcoma. As acidic pH inhibits anti-tumor activities of NK cells, we sought to determine if there was a similar reduction in the anticryptococcal activity of NK cells. Surprisingly, we found that both primary human NK cells and the human NK cell line, YT, have preserved or even enhanced killing of Cryptococcus in acidic, compared to physiological, pH. Studies to explore the mechanism of enhanced killing revealed that acidic pH does not increase the effector to target ratio, binding of cytolytic cells to Cryptococcus, or the active perforin content in effector cells. By contrast, perforin degranulation was greater at acidic pH, and increased degranulation was preceded by enhanced ERK1/2 phosphorylation, which is essential for killing. Moreover, using a replication defective ras1 knockout strain of Cryptococcus increased degranulation occurred during more rapid replication of the organisms. Finally, NK cells were found intimately associated with C. gattii within the cryptococcoma of a fatal infection. These results suggest that NK cells have amplified signaling, degranulation, and greater killing at low pH and when the organisms are replicating quickly, which would help maintain microbicidal host defense despite an acidic microenvironment.

Liver X receptor ß is essential for the differentiation of radial glial cells to oligodendrocytes in the dorsal cortex.

Several psychiatric disorders are associated with aberrant white matter development, suggesting oligodendrocyte and myelin dysfunction in these diseases. There are indications that radial glial cells (RGCs) are involved in initiating myelination, and may contribute to the production of oligodendrocyte progenitor cells (OPCs) in the dorsal cortex. Liver X receptors (LXRs) are involved in maintaining normal myelin in the central nervous system (CNS), however, their function in oligodendrogenesis and myelination is not well understood. Here, we demonstrate that loss of LXRß function leads to abnormality in locomotor activity and exploratory behavior, signs of anxiety and hypomyelination in the corpus callosum and optic nerve, providing in vivo evidence that LXRß deletion delays both oligodendrocyte differentiation and maturation. Remarkably, along the germinal ventricular zone-subventricular zone and corpus callosum there is reduced OPC production from RGCs in LXRß(-/-) mice. Conversely, in cultured RGC an LXR agonist led to increased differentiation into OPCs. Collectively, these results suggest that LXRß, by driving RGCs to become OPCs in the dorsal cortex, is critical for white matter development and CNS myelination, and point to the involvement of LXRß in psychiatric disorders.

In vitro and in vivo evidence for amphotericin B as a P-glycoprotein substrate on the blood-brain barrier.

Amphotericin B (AMB) has been a mainstay therapy for fungal infections of the central nervous system, but its use has been limited by its poor penetration into the brain, the mechanism of which remains unclear. In this study, we aimed to investigate the role of P-glycoprotein (P-gp) in AMB crossing the blood-brain barrier (BBB). The uptake of AMB by primary brain capillary endothelial cells in vitro was significantly enhanced after inhibition of P-gp by verapamil. The impact of two model P-gp inhibitors, verapamil and itraconazole, on brain/plasma ratios of AMB was examined in both uninfected CD-1 mice and those intracerebrally infected with Cryptococcus neoformans. In uninfected mice, the brain/plasma ratios of AMB were increased 15 min (3.5 versus 2.0; P < 0.05) and 30 min (5.2 versus 2.8; P < 0.05) after administration of verapamil or 45 min (6.0 versus 3.9; P < 0.05) and 60 min (5.4 versus 3.8; P < 0.05) after itraconazole administration. The increases in brain/plasma ratios were also observed in infected mice treated with AMB and P-gp inhibitors. The brain tissue fungal CFU in infected mice were significantly lower in AMB-plus-itraconazole or verapamil groups than in the untreated group (P < 0.005), but none of the treatments protected the mice from succumbing to the infection. In conclusion, we demonstrated that P-gp inhibitors can enhance the uptake of AMB through the BBB, suggesting that AMB is a P-gp substrate.

Immunohistological detection of Chlamydia pneumoniae in the Alzheimer's disease brain.

BACKGROUND: Sporadic late-onset Alzheimer's disease (AD) appears to evolve from an interplay between genetic and environmental factors. One environmental factor that continues to be of great interest is that of Chlamydia pneumoniae infection and its association with late-onset disease. Detection of this organism in clinical and autopsy samples has proved challenging using a variety of molecular and histological techniques. Our current investigation utilized immunohistochemistry with a battery of commercially available anti-C. pneumoniae antibodies to determine whether C. pneumoniae was present in areas typically associated with AD neuropathology from 5 AD and 5 non-AD control brains. RESULTS: Immunoreactivity for C. pneumoniae antigens was observed both intracellularly in neurons, neuroglia, endothelial cells, and peri-endothelial cells, and extracellularly in the frontal and temporal cortices of the AD brain with multiple C. pneumoniae-specific antibodies. This immunoreactivity was seen in regions of amyloid deposition as revealed by immunolabeling with two different anti-beta amyloid antibodies. Thioflavin S staining, overlaid with C. pneumoniae immunolabeling, demonstrated no direct co-localization of the organism and amyloid plaques. Further, the specificity of C. pneumoniae labeling of AD brain sections was demonstrated using C. pneumoniae antibodies pre-absorbed against amyloid ß 1-40 and 1-42 peptides. CONCLUSIONS: Anti-C. pneumoniae antibodies, obtained commercially, identified both typical intracellular and atypical extracellular C. pneumoniae antigens in frontal and temporal cortices of the AD brain. C. pneumoniae, amyloid deposits, and neurofibrillary tangles were present in the same regions of the brain in apposition to one another. Although additional studies are required to conclusively characterize the nature of Chlamydial immunoreactivity in the AD brain, these results further implicate C. pneumoniae infection with the pathogenesis of Alzheimer's disease.

HTLV-III infection in brains of children and adults with AIDS encephalopathy.

Unexplained debilitating dementia or encephalopathy occurs frequently in adults and children with the acquired immune deficiency syndrome (AIDS). Brains from 15 individuals with AIDS and encephalopathy were examined by Southern analysis and in situ hybridization for the presence of human T-cell leukemia (lymphotropic) virus type III (HTLV-III), the virus believed to be the causative agent of AIDS. HTLV-III DNA was detected in the brains of five patients, and viral-specific RNA was detected in four of these. In view of these findings and the recent demonstration of morphologic and genetic relatedness between HTLV-III and visna virus, a lentivirus that causes a chronic degenerative neurologic disease in sheep, HTLV-III should be evaluated further as a possible cause of AIDS encephalopathy.

Transneuronal transfer of herpes virus from peripheral nerves to cortex and brainstem.

The transneuronal transfer of neurotropic viruses may represent an effective tool for tracing chains of connected neurons because replication of virus in the recipient neurons after transfer amplifies the "tracer signal." Herpes simplex virus type 1 was transferred transneuronally from forelimb and hindlimb nerves of rats to the cortical and brainstem neurons that project to the spinal enlargements to which the nerves receiving injections are connected. This transneuronal transfer of herpes simplex virus type 1 from peripheral nerves has the potential to be used to identify neurons in the brain that are related transsynaptically to different nerves and muscles.

Amoebic meningoencephalitis: 16 fatalities.

The parasitologic examination of pathologic brain tissue from 16 cases of acute plurulent meningoencephalitis occurring between 1962 and 1965 in northern Bohemia disclosed massive infection of the central nervous system by amoebas of the limax type. The common source was an indoor swimming pool.

Mycotic encephalitis and nephritis in a dog due to infection with Cladosporium cladosporioides.

The dematiaceous fungus Cladosporium cladosporioides is a widely distributed saprophyte that is reported to occasionally infect the lung, skin, eye and brain of humans. This report describes a German shepherd dog with granulomatous encephalitis and nephritis due to C. cladosporioides infection. Although the fungal organisms appeared non-pigmented in haematoxylin and eosin stained sections, they were readily identified with histochemical stains. Semi-nested polymerase chain reaction using universal fungal primers amplified fungal DNA from fixed tissue that had identity to that of C. cladosporioides on sequencing.

Cortical recovery of swallowing function in wound botulism.

BACKGROUND: Botulism is a rare disease caused by intoxication leading to muscle weakness and rapidly progressive dysphagia. With adequate therapy signs of recovery can be observed within several days. In the last few years, brain imaging studies carried out in healthy subjects showed activation of the sensorimotor cortex and the insula during volitional swallowing. However, little is known about cortical changes and compensation mechanisms accompanying swallowing pathology. METHODS: In this study, we applied whole-head magnetoencephalography (MEG) in order to study changes in cortical activation in a 27-year-old patient suffering from wound botulism during recovery from dysphagia. An age-matched group of healthy subjects served as control group. A self-paced swallowing paradigm was performed and data were analyzed using synthetic aperture magnetometry (SAM). RESULTS: The first MEG measurement, carried out when the patient still demonstrated severe dysphagia, revealed strongly decreased activation of the somatosensory cortex but a strong activation of the right insula and marked recruitment of the left posterior parietal cortex (PPC). In the second measurement performed five days later after clinical recovery from dysphagia we found a decreased activation in these two areas and a bilateral cortical activation of the primary and secondary sensorimotor cortex comparable to the results seen in a healthy control group. CONCLUSION: These findings indicate parallel development to normalization of swallowing related cortical activation and clinical recovery from dysphagia and highlight the importance of the insula and the PPC for the central coordination of swallowing. The results suggest that MEG examination of swallowing can reflect short-term changes in patients suffering from neurogenic dysphagia.

A novel cerebral microangiopathy with endothelial cell atypia and multifocal white matter lesions: a direct mycoplasmal infection?

We present 3 sporadic cases of a subacute to chronic, progressive motor (i.e. weakness, ataxia, spasticity, dysarthria, and dysphagia) and cognitive disorder in adults of both sexes, without proven immunocompromise or malignancy. Neuroimaging studies revealed tiny calcifications with atrophy of the cerebrum, pons, and midbrain in 1 patient, cerebral atrophy in another, and cerebral atrophy and periventricular white matter hyperintensities in the third. Clinical diagnoses included cortico-pontine-cerebellar degeneration, mixed neurodegenerative disorder, progressive supranuclear palsy, diffuse Lewy body disease, and Lyme disease. One atrophic brain revealed widely disseminated, millimeter-sized gray lesions in cerebral white matter and obscured anatomic markings of the basis pontis. The most conspicuous microscopic feature in all was capillaries with focally piled up endothelial nuclei, some of which appeared to be multinucleated, or enlarged, hyperchromatic crescentic single nuclei. Although seen mostly without associated damage, they were also noted with white matter lesions displaying vacuolation, demyelination, spheroids, necrosis, vascular fibrosis, and mineralization; these were most severe in the basis pontis. Immunostains and probes to herpes simplex virus-I, -II, and -8; adenovirus, cytomegalovirus, varicella-zoster, Epstein-Barr virus, measles, JC virus, and herpes hominis virus-6 were negative. Electron microscopy revealed no virions in endothelial cells with multilobed or multiple nuclei and duplicated basal laminae. However, mycoplasma-like bodies, mostly 400 to 600 nm in size, were found in endothelial cell cytoplasm and capillary lumina. Platelets adhered to affected endothelial cells. Polymerase chain reaction and immunohistochemistry of fixed samples for Mycoplasma fermentans were negative; other species of Mycoplasma remain viable pathogenic candidates.

Fungal infections of the central nervous system: the clinical syndromes.

Fungal infections of the central nervous system (CNS) are being increasingly diagnosed both in immunocompromised and immunocompetent individuals. Sinocranial aspergillosis is more frequently described from countries with temperate climates, more often in otherwise immunocompetent individuals. The clinical syndromes with which fungal infections of the CNS can present are protean and can involve most part of the neuroaxis. Certain clinical syndromes are specific for certain fungal infections. The rhinocerebral form is the most common presenting syndrome with zygomycosis and skull-base syndromes are often the presenting clinical syndromes in patients with sinocranial aspergillosis. Subacute and chronic meningitis in patients with HIV infection is more likely to be due to cryptococcal infection. Early recognition of the clinical syndromes in an appropriate clinical setting is the first step towards achieving total cure in some of these infections.

Circulating bacterial lipopolysaccharide-induced inflammation reduces flow in brain-irrigating arteries independently from cerebrovascular prostaglandin production.

Brain dysfunction is a frequent complication of the systemic inflammatory response to bacterial infection or sepsis. In the present work, the effects of intravenous bacterial lipopolysaccharide (LPS) administration on cerebral arterial blood flow were assessed with time-of-flight (TOF)-based magnetic resonance angiography (MRA) in mice. Cerebral expression of the transcription factors nuclear factor-kappaB (NF-κB) and c-Fos and that of enzymes synthesizing vasoactive mediators, such as prostaglandins and nitric oxide, known to be increased under inflammatory conditions, were studied in the same animals. Time-resolved TOF MRA revealed no differences in blood flow in the internal carotids upstream of the circle of Willis, but indicated lower flow in its lateral parts as well as in the middle and anterior cerebral arteries after intravenous LPS injection as compared to saline administration. Although LPS did not increase c-Fos expression in ventral forebrain structures of these animals, it did induce NF-κB in meningeal blood vessels. LPS also increased cerebral expression of cyclooxygenase-2 and prostaglandin E synthase mRNAs, but de novo expression occurred in veins rather than in arteries. In conclusion, our work indicates that LPS-induced systemic inflammation does not necessarily affect filling of the circle of the Willis from the periphery, but that circulating LPS alters outflow from the circle of Willis to the middle and anterior cerebral arteries. These modifications in arterial flow were not related to increased cerebral synthesis of prostaglandins, but may instead be the consequence of the action of circulating prostaglandins and other vasoactive mediators on brain-irrigating arteries during systemic inflammation.

Survival of rat cerebrocortical neurons after rickettsial infection.

Neuroinvasive microorganisms are suspected to play an important role in the etiopathogenesis of neurological diseases. However, direct evidence for the pathogenic function is still missing. The main aim of this study was to investigate biochemical and morphological changes that may occur as a result of an in vitro infection of rat cerebrocortical neurons by selected members of the genus Rickettsia. Our results showed that survival of the neurons is significantly reduced after the infection. Intracellular level of ATP is gradually decreased and inversely correlates with the load of rickettsiae. Immunofluorescence revealed that rickettsiae can enter the neurons and are localized in perinuclear space and also in neuronal processes. Data obtained in this study correspond to the idea of possible involvement of rickettsiae in the etiopathogenesis of various neuropathies.

Detection of HIV proviral DNA in cortex and white matter of AIDS brains by non-isotopic polymerase chain reaction: correlation with diffuse poliodystrophy.

OBJECTIVE: (1) To determine whether detection of HIV proviral DNA sequences in the cerebral cortex correlates with the presence of pathological changes in this region, believed to contribute to the HIV-associated cognitive/motor complex. (2) To compare the frequency with which HIV infects cortical and subcortical regions of the brain. DESIGN: In vitro studies on HIV neurotoxicity suggest that HIV may be involved in the pathogenesis of cortical damage, recently defined as diffuse poliodystrophy (DPD) in AIDS. Previous detection of HIV antigen has localized HIV more frequently to subcortical than to cortical regions. It is not known whether HIV preferentially infects subcortical tissues or if viral expression varies in these two regions. METHODS: HIV antigen and proviral DNA sequences were detected in anterior frontal lobe tissues using immunohistochemistry (IHC) and the polymerase chain reaction (PCR), respectively. DPD was assessed by staining with antibodies against astrocytes (GFAP) and microglia/macrophages (HAM 56). RESULTS: HIV proviral DNA was detected in nine out of 15 cortical samples and in 10 out of 15 white matter samples, whilst HIV p24 antigen was localized to the cortex in three out of 15 and to the white matter in seven out of 15 cases. DPD was found in 10 cases, although in five a different aetiology may have been involved. However, DPD was present in eight out of the nine cases in which HIV proviral DNA was detected in the cortex. CONCLUSIONS: Using a non-isotopic PCR method, HIV was detected in the brains of more cases than would be expected on the basis of IHC detection, and was present in the cortex as frequently as in the white matter. HIV, together with other factors, may contribute to the pathogenesis of DPD.

Experimental panencephalitis induced in suckling mice, by parainfluenza type 1 (6/94) virus, II. Virologic studies.

6/94 virus, a parainfluenza type 1 isolate from multiple sclerosis brain tissue, produced a chronic panencephalitis when inoculated intracerebrally into suckling ICR mice. Immunofluorescent staining revealed 6/94 viral antigen in ependyma, meninges, choroid plexus, and perivascular parenchymal sites from day 3 to 128 days after infection. Hemadsorption-neutralizing antibody was first detected between 20-25 days after infection and remained at high titers for 7 months. Using embryonated chicken eggs, virus was recovered from mouse brains for only 8 days, but could be recovered from brains grown in vitro as explants for 37 days after infection. In cell lines established from explanted brain tissue, immunofluorescence was the most sensitive indicator of virus presence, although infectious virus was not produced. Fusion of these mouse brain cells with human (W138) indicator cells was the most effective means of rescuing 6/94 virus.

Pathogenesis and pathology of scrapie after stereotactic injection of strain 22L in intact and bisected cerebella.

The mechanisms involved in the spread of scrapie within the brain remain unclear. To examine this issue the 22L scrapie strain was injected in one side of the cerebellum of mice in which the cerebellum had been bisected prior to injection. Another group of animals received the same injection into intact cerebella, i.e. without prior bisection. We found that bisection of the cerebella delayed the spread of scrapie agent from the injection site to the contralateral side of the cerebellum and that the occurrence of vacuolization was not as extensive and was markedly delayed in the uninjected side compared to its occurrence after injection in the intact cerebellum. Replication of agent in an area preceded the development of vacuolization in that area by several weeks. There was marked loss of Purkinje cells on the injected side of bisected cerebella, with no loss seen on the uninjected side. The incubation period of scrapie disease in mice injected after cerebellar bisection was significantly longer than after the injection of intact cerebella. The results in this study suggest that the scrapie agent spreads along intact nerve cell tracts, probably by axonal transport.