RESUMO
Canine degenerative myelopathy (DM) is a progressive neurological disorder that may be considered to be a large animal model for specific forms of the fatal human disease, familial amyotrophic lateral sclerosis (fALS). DM is associated with a c118G>A mutation of the superoxide dismutase 1 (Sod1) gene, and a significant proportion of cases are inherited in an autosomal recessive manner in contrast to the largely, but not exclusively, dominant mode of inheritance in fALS. The consensus view is that these Sod1/SOD1 mutations result in a toxic gain of function but the mechanisms remain unclear. Here we used an in vitro neuroblastoma cell line transfection system to monitor wild-type and mutant forms of SOD1 fusion proteins containing either a Cherry or an enhanced green fluorescent protein (EGFP) tag. These fusion proteins retained SOD1 enzymatic activity on a native gel assay system. We demonstrate that SOD1 aggregate density is significantly higher in DM transfectants compared to wild-type. In addition, we show by co-immunoprecipitation and confocal microscopy, evidence for a potential interaction between wild-type and mutant forms of SOD1 in co-transfected cells. While in vitro studies have shown SOD1 heterodimer formation in fALS models, this is the first report for DM SOD1. Therefore, despite for the majority of cases there is a difference in the mode of inheritance between fALS and DM, a similar interaction between wild-type and mutant SOD1 forms can occur. Clarifying the role of SOD1 in DM may also be of benefit to understanding the role of SOD1 in fALS.
Assuntos
Esclerose Lateral Amiotrófica/genética , Mutação/genética , Superóxido Dismutase-1/genética , Superóxido Dismutase/genética , Animais , Linhagem Celular , Modelos Animais de Doenças , Cães , Proteínas de Fluorescência Verde/genética , Humanos , Doenças da Medula Espinal/genéticaRESUMO
BACKGROUND: Nasal secretion (NS) was investigated as a source of information regarding the mucosal and systemic immune status of cattle challenged by respiratory disease. A method for the collection of substantial volumes (~12 ml) of NS from cattle was developed to establish a reference range of analytes that are present in the NS of healthy cattle. Biochemical profiles of NS from a group of 38 healthy Holstein-Friesian cows revealed high alkaline phosphatase (AP) activity of up to 2392 IU/L. The character and source of the high activity of AP in bovine NS was investigated. RESULTS: Histochemical analysis confirmed the localization of the AP enzyme activity to epithelial cells and serous glands of the nasal respiratory mucosa. Analysis of mRNA levels from nasal mucosa by end point RT-PCR and PCR product sequencing confirmed that the AP was locally produced and is identical at the nucleotide level to the non-specific AP splice variant found in bovine liver, bone and kidney. Analysis by isoelectric focussing confirmed that AP was produced locally at a high level in nasal epithelium demonstrating that AP from nasal secretion and nasal mucosa had similar pI bands, though differing from those of the liver, kidney, bone and intestine, suggesting different post-translational modification (PTM) of AP in these tissues. CONCLUSIONS: A nasal isozyme of AP has been identified that is present at a high activity in NS, resulting from local production and showing distinctive PTM and may be active in NS as an anti-endotoxin mediator.
Assuntos
Fosfatase Alcalina/análise , Bovinos/metabolismo , Mucosa Nasal/metabolismo , Fosfatase Alcalina/genética , Animais , Secreções Corporais/enzimologia , Feminino , Focalização Isoelétrica/veterinária , Mucosa Nasal/enzimologia , Isoformas de Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterináriaRESUMO
Varicella-Zoster virus (VZV) is a pathogenic human herpes virus that causes varicella ("chicken pox") as a primary infection, following which it becomes latent in neuronal cells in human peripheral ganglia. It may then reactivate to cause herpes zoster ("shingles"). Defining the pattern of VZV gene expression during latency is an important issue, and four highly expressed VZV genes were first identified by Randall Cohrs in 1996 using cDNA libraries. Further studies from both his and other laboratories, including our own, have suggested that viral gene expression may be more widespread than previously thought, but a confounding factor has always been the possibility of viral reactivation after death in tissues obtained even at 24 h post-mortem. Recent important studies, which Randall Cohrs contributed to, have clarified this issue by studying human trigeminal ganglia at 6 h after death using RNA-Seq methodology when a novel spliced latency-associated VZV transcript (VLT) was found to be mapped antisense to the viral transactivator gene 61. Viral gene expression could be induced by a VLT-ORF 63 fusion transcript when VZV reactivated from latency. Prior detection by several groups of ORF63 in post-mortem-acquired TG is very likely to reflect detection of the VLT-ORF63 fusion and not canonical ORF63. The contributions to the VZV latency field by Randall Cohrs have been numerous and highly significant.
Assuntos
Varicela , Herpes Zoster , Gânglios , Expressão Gênica , Herpes Zoster/patologia , Herpesvirus Humano 3/genética , Humanos , Latência Viral/genéticaRESUMO
Zika virus (ZIKV) is a neurotropic flavivirus recently linked to congenital ZIKV syndrome in children and encephalitis and Guillain-Barré syndrome in adults. Neurotropic viruses often use axons to traffic to neuronal or glial cell somas where they either remain latent or replicate and proceed to infect new cells. Consequently, it has been suggested that axon degeneration could represent an evolutionarily conserved mechanism to limit viral spread. Whilst it is not known if ZIKV transits in axons, we previously reported that ZIKV infection of glial cells in a murine spinal cord-derived cell culture model of the CNS is associated with a profound loss of neuronal cell processes. This, despite that postmitotic neurons are relatively refractory to infection and death. Here, we tested the hypothesis that ZIKV-associated degeneration of neuronal processes is dependent on activation of Sterile alpha and armadillo motif-containing protein 1 (SARM1), an NADase that acts as a central executioner in a conserved axon degeneration pathway. To test this, we infected wild type and Sarm1 homozygous or heterozygous null cell cultures with ZIKV and examined NAD+ levels as well as the survival of neurons and their processes. Unexpectedly, ZIKV infection led to a rapid SARM1-independent reduction in NAD+. Nonetheless, the subsequent profound loss of neuronal cell processes was SARM1-dependent and was preceded by early changes in the appearance of ß-tubulin III staining. Together, these data identify a role for SARM1 in the pathogenesis of ZIKV infection, which may reflect SARM1's conserved prodegenerative function, independent of its NADase activity.
RESUMO
Human African trypanosomiasis (HAT), also known as sleeping sickness, is a major cause of mortality and morbidity in sub-Saharan Africa. We hypothesised that recent findings of neurological features and parasite brain infiltration occurring at much earlier stages in HAT than previously thought could be explained by early activation of host genetic programmes controlling CNS disease. Accordingly, a transcriptomal analysis was performed on brain tissue at 0, 7, 14, 21 and 28dpi from the HAT CD1/GVR35 mouse model. Up to 21dpi, most parasites are restricted to the blood and lymphatic system. Thereafter the trypanosomes enter the brain initiating the encephalitic stage. Analysis of ten different time point Comparison pairings, revealed a dynamic transcriptome comprising four message populations. All 7dpi Comparisons had by far more differentially expressed genes compared to all others. Prior to invasion of the parenchyma, by 7dpi, ~2,000 genes were up-regulated, denoted [7dpi↑] in contrast to a down regulated population [7dpi↓] also numbering ~2,000. However, by 14dpi both patterns had returned to around the pre-infected levels. The third, [28dpi↑] featured over three hundred transcripts which had increased modestly up to14dpi, thereafter were significantly up-regulated and peaked at 28dpi. The fourth, a minor population, [7dpi↑-28dpi↑], had similar elevated levels at 7dpi and 28dpi. KEGG and GO enrichment analysis predicted a diverse phenotype by 7dpi with changes to innate and adaptive immunity, a Type I interferon response, neurotransmission, synaptic plasticity, pleiotropic signalling, circadian activity and vascular permeability without disruption of the blood brain barrier. This key observation is consistent with recent rodent model neuroinvasion studies and clinical reports of Stage 1 HAT patients exhibiting CNS symptoms. Together, these findings challenge the strict Stage1/Stage2 phenotypic demarcation in HAT and show that that significant neurological, and immune changes can be detected prior to the onset of CNS disease.
Assuntos
Encéfalo/parasitologia , Doenças do Sistema Nervoso Central/genética , Doenças do Sistema Nervoso Central/imunologia , Trypanosoma brucei brucei/fisiologia , Tripanossomíase Africana/genética , Tripanossomíase Africana/imunologia , Animais , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/parasitologia , Encéfalo/imunologia , Doenças do Sistema Nervoso Central/parasitologia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Fenótipo , Análise Serial de Proteínas , Transcrição Gênica , Tripanossomíase Africana/parasitologiaRESUMO
The most common cause of Pelizaeus-Merzbacher (PMD) is due to duplication of the PLP1 gene but it is unclear how increased gene dosage affects PLP turnover and causes dysmyelination. We have studied the dynamics of PLP/DM20 in a transgenic mouse model of PMD with increased gene dosage of the proteolipid protein gene (Plp1). The turnover of PLP/DM20 were investigated using an ex-vivo brain slice system and cultured oligodendrocytes. Homozygous mice have reduced PLP translation, markedly enhanced PLP degradation, and markedly reduced incorporation of PLP into myelin. Proteasome inhibition (MG132) prevented the enhanced degradation. Numerous autophagic vesicles are present in homozygous transgenic mice that may influence protein dynamics. Surprisingly, promoting autophagy with rapamycin decreases the degradation of nascent PLP suggesting autophagic vacuoles serve as a cellular storage compartment. We suggest that there are multiple subcellular fates of PLP/DM20 when overexpressed: the vast majority being degraded by the proteasome, a proportion sequestered into autophagic vacuoles, probably fused with endolysosomes, and only a small proportion entering the myelin sheath, where its association with lipid rafts is perturbed. Transgenic oligodendrocytes have fewer membrane sheets and this phenotype is improved with siRNA-mediated knockdown of PLP expression that promotes the formation of MBP+ myelin-like sheets. This finding suggests that RNAi technology is in principle applicable to improve CNS myelination when compromised by PLP/DM20 overexpression.
Assuntos
Predisposição Genética para Doença/genética , Proteína Proteolipídica de Mielina/genética , Bainha de Mielina/genética , Bainha de Mielina/metabolismo , Doença de Pelizaeus-Merzbacher/genética , Doença de Pelizaeus-Merzbacher/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Proteolipídica de Mielina/antagonistas & inibidores , Proteína Proteolipídica de Mielina/biossíntese , Técnicas de Cultura de Órgãos , Interferência de RNA/fisiologia , Vacúolos/metabolismo , Vacúolos/ultraestruturaRESUMO
The rumpshaker mutation of the murine myelin proteolipid protein 1 (Plp1) gene generates misfolded PLP/DM20 protein, resulting in dysmyelination, increased oligodendrocyte apoptosis, and death prior to P40 when expressed on the C57 BL/6 background. In this study, we used transgenic complementation to normalize the levels of PLP/DM20 in myelin with wild-type protein to determine whether loss of normal PLP function or gain of toxic function is responsible for dysmyelination in the rumpshaker. Restoring myelin PLP/DM20 levels extended the survival time to at least P60, significantly reduced the density of apoptotic cells, increased myelin volume, and restored normal periodicity of myelin. Biochemical analysis found that several myelin proteins that are reduced in rumpshaker, including MAG, CNP, and SirT2, are markedly elevated at peak myelination (P20) in the rumpshaker transgenic mouse. Myelin basic protein, however, remained low at peak myelination but was restored at P60 when myelin had matured and entered into a maintenance phase. Markers of the unfolded protein response (UPR), BiP and XBP1, remained activated with the introduction of wild-type PLP. These data demonstrate that restoring wild-type PLP/DM20 levels in rumpshaker improves the phenotype and the integrity of myelin, but hypomyelination persists and stress pathways remain activated. This suggests that both gain- and loss-of-function mechanisms are involved in the pathogenesis of the rumpshaker.
Assuntos
Apoptose/fisiologia , Proteína Proteolipídica de Mielina/metabolismo , Bainha de Mielina/metabolismo , Fenótipo , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Astrócitos/patologia , Astrócitos/fisiologia , Proteínas de Ligação a DNA/metabolismo , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Básica da Mielina , Proteína Proteolipídica de Mielina/genética , Bainha de Mielina/patologia , Glicoproteína Associada a Mielina , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/metabolismo , Fatores de Transcrição de Fator Regulador X , Sirtuína 2/metabolismo , Medula Espinal/metabolismo , Análise de Sobrevida , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Proteína 1 de Ligação a X-BoxRESUMO
BACKGROUND: Process formation by glial cells is crucial to their function. Mayven, an actin binding, multi-domain polypeptide, and member of the BTB-BACK-Kelch family have been shown to be important in oligodendrocyte process extension. To assess the role of Mayven in neural cell process extension we have tracked the subcellular distribution of exogenous Mayven following expression of a rat Mayven -EGFP cDNA in a variety of neural cell backgrounds and specifically in OEC tranfectants following drug treatment to disrupt the integrity of the cytoskeleton. A comparison was made between the subcellular localization following transient transfection of OECs with full-length Mayven cDNA and a series of mutant domain constructs. RESULTS: The subcellular location of Mayven in OEC transfectants showed a characteristic distribution with intense foci of staining towards the process tips corresponding to regions of accumulated Mayven overlapping in part with lammelipodial actin and was absent from the filipodia and the outer membrane. This signature pattern was also observed in Schwann cells, Oli-Neu cells, astrocytes and the neuroblastoma cell line B104 transfectants and resembled the exogenous and endogenous Mayven distribution in oligodendrocytes. This contrasted with the localization pattern in non-neural cells. There was a re-localization of Mayven in OEC transfectants following drug treatment to challenge the integrity of the actin cytoskeleton while breakdown of the microtubular component had no discernible impact on the accumulation of Mayven in the process tips. Deletion of the first three amino acids of the SH3 motif of the putative Fyn Kinase binding domain at the amino terminus significantly compromised this signature pattern as did the removal of the last Kelch repeat unit of six unit Kelch domain comprising the carboxyl terminus. In addition, there was a reduction in process length in mutant transfectants. Co-expression studies with a haemagglutinin (HA) tagged wild type Mayven cDNA and EGFP tagged mutant cDNAs suggested a homomeric interaction mediated by the BTB/POZ domain. CONCLUSIONS: Exogenous Mayven is transported to the lamellipodia in neural transfectants associating with the actin cytoskeletal network. In addition to the importance of the internal BTB/POZ domain, this subcellular distribution pattern is dependent on the presence of an intact amino and carboxyl terminus.
Assuntos
Citoesqueleto/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Citoesqueleto/genética , DNA Complementar/genética , DNA Complementar/metabolismo , Imuno-Histoquímica , Proteínas dos Microfilamentos/genética , Microscopia Confocal , Proteínas do Tecido Nervoso/genética , Ratos , TransfecçãoRESUMO
Varicella-Zoster virus (VZV) is a human herpes virus that reactivates from a latent state in human trigeminal and dorsal root ganglia to cause herpes zoster (shingles) which is a painful vesicular dermatomal skin eruption. The major complication of herpes zoster is post-herpetic neuralgia (PHN) which is a serious condition occurring especially in individuals over 50 years. PHN is extremely painful, may be permanent, and is frequently very refractory to all treatment. The ability to identify those patients with herpes zoster who are likely to develop PHN would be highly beneficial as it would allow pre-emptive anti-viral therapy. We have assessed the potential of using long oligonucleotide VZV microarrays to determine whether MeWo cells infected with VZV isolates obtained from 13 patients with zoster who had subsequently developed PHN showed significant transcriptomal differences from MeWo cells infected with viruses isolated from ten zoster patients who had not developed PHN. We found that viral gene expression from sample to sample within a group (PHN patients or non-PHN patients) varied as much, or more, than the viral gene expression between those groups. Quantitative real-time polymerase chain reaction studies carried out on 11 open reading frames on four representative viral infected MeWo cell lines (two from each group) confirmed the transcriptomal heterogeneity between the two groups. Growth curve analyses of ten representative infected cell lines (five from each group) showed that PHN and non-PHN-associated viruses replicated equally efficiently. Taken together, these findings suggest that viral microarray-based transcriptomal measurements are unlikely to prove of clinical utility in predicting the incidence of PHN.
Assuntos
Perfilação da Expressão Gênica , Herpes Zoster/virologia , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/patogenicidade , Neuralgia Pós-Herpética/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular , Feminino , Herpesvirus Humano 3/isolamento & purificação , Humanos , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Oligodendrocytes are critical for the development of the plasma membrane and cytoskeleton of the axon. In this paper, we show that fast axonal transport is also dependent on the oligodendrocyte. Using a mouse model of hereditary spastic paraplegia type 2 due to a null mutation of the myelin Plp gene, we find a progressive impairment in fast retrograde and anterograde transport. Increased levels of retrograde motor protein subunits are associated with accumulation of membranous organelles distal to nodal complexes. Using cell transplantation, we show categorically that the axonal phenotype is related to the presence of the overlying Plp null myelin. Our data demonstrate a novel role for oligodendrocytes in the local regulation of axonal function and have implications for the axonal loss associated with secondary progressive multiple sclerosis.
Assuntos
Axônios/metabolismo , Oligodendroglia/metabolismo , Paraplegia Espástica Hereditária/patologia , Alelos , Animais , Transporte Biológico , Western Blotting , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Modelos Animais de Doenças , Heterozigoto , Imuno-Histoquímica , Camundongos , Camundongos Mutantes , Bainha de Mielina/metabolismo , Nervo Óptico/metabolismo , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/metabolismo , Medula Espinal/patologia , Fatores de TempoRESUMO
After injury, the CNS undergoes an astrocyte stress response characterized by reactive astrocytosis/proliferation, boundary formation, and increased glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycan (CSPG) expression. Previously, we showed that in vitro astrocytes exhibit this stress response when in contact with Schwann cells but not olfactory ensheathing cells (OECs). In this study, we confirm this finding in vivo by demonstrating that astrocytes mingle with OECs but not Schwann cells after injection into normal spinal cord. We show that Schwann cell-conditioned media (SCM) induces proliferation in monocultures of astrocytes and increases CSPG expression in a fibroblast growth factor receptor 1 (FGFR1)-independent manner. However, SCM added to OEC/astrocyte cocultures induces reactive astrocytosis and boundary formation, which, although sensitive to FGFR1 inhibition, was not induced by FGF2 alone. Addition of heparin to OEC/astrocyte cultures induces boundary formation, whereas heparinase or chlorate treatment of Schwann cell/astrocyte cultures reduces it, suggesting that heparan sulfate proteoglycans (HSPGs) are modulating this activity. In vivo, FGF2 and FGFR1 immunoreactivity was increased over grafted OECs and Schwann cells compared with the surrounding tissue, and HSPG immunoreactivity is increased over reactive astrocytes bordering the Schwann cell graft. These data suggest that components of the astrocyte stress response, including boundary formation, astrocyte hypertrophy, and GFAP expression, are mediated by an FGF family member, whereas proliferation and CSPG expression are not. Furthermore, after cell transplantation, HSPGs may be important for mediating the stress response in astrocytes via FGF2. Identification of factors secreted by Schwann cells that induce this negative response in astrocytes would further our ability to manipulate the inhibitory environment induced after injury to promote regeneration.
Assuntos
Astrócitos/metabolismo , Comunicação Celular/fisiologia , Fatores de Crescimento de Fibroblastos/farmacologia , Gliose/metabolismo , Heparina/farmacologia , Bulbo Olfatório/metabolismo , Células de Schwann/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Bulbo Olfatório/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Células de Schwann/efeitos dos fármacosRESUMO
BACKGROUND: Human African trypanosomiasis or sleeping sickness, caused by the parasite Trypanosoma brucei, leads to neuroinflammation and characteristic sleep/wake alterations. The relationship between the onset of these alterations and the development of neuroinflammation is of high translational relevance, but remains unclear. This study investigates the expression of interferon (IFN)-γ and IFN-inducible chemokine genes in the brain, and the levels of CXCL10 in the serum and cerebrospinal fluid prior to and during the encephalitic stage of trypanosome infection, and correlates these with sleep/wake changes in a rat model of the disease. METHODOLOGY/PRINCIPAL FINDINGS: The expression of genes encoding IFN-γ, CXCL9, CXCL10, and CXCL11 was assessed in the brain of rats infected with Trypanosoma brucei brucei and matched controls using semi-quantitative end-point RT-PCR. Levels of CXCL10 in the serum and cerebrospinal fluid were determined using ELISA. Sleep/wake states were monitored by telemetric recording. Using immunohistochemistry, parasites were found in the brain parenchyma at 14 days post-infection (dpi), but not at 6 dpi. Ifn-γ, Cxcl9, Cxcl10 and Cxcl11 mRNA levels showed moderate upregulation by 14 dpi followed by further increase between 14 and 21 dpi. CXCL10 concentration in the cerebrospinal fluid increased between 14 and 21 dpi, preceded by a rise in the serum CXCL10 level between 6 and 14 dpi. Sleep/wake pattern fragmentation was evident at 14 dpi, especially in the phase of wake predominance, with intrusion of sleep episodes into wakefulness. CONCLUSIONS/SIGNIFICANCE: The results show a modest increase in Cxcl9 and Cxcl11 transcripts in the brain and the emergence of sleep/wake cycle fragmentation in the initial encephalitic stage, followed by increases in Ifn-γ and IFN-dependent chemokine transcripts in the brain and of CXCL10 in the cerebrospinal fluid. The latter parameter and sleep/wake alterations could provide combined humoral and functional biomarkers of the early encephalitic stage in African trypanosomiasis.
Assuntos
Quimiocinas/sangue , Quimiocinas/líquido cefalorraquidiano , Encefalite/parasitologia , Sono , Tripanossomíase Africana/sangue , Tripanossomíase Africana/líquido cefalorraquidiano , Animais , Biomarcadores , Encéfalo/parasitologia , Encéfalo/patologia , Interferon gama/sangue , Interferon gama/líquido cefalorraquidiano , Masculino , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Análise de Regressão , Trypanosoma brucei bruceiRESUMO
BACKGROUND: The timing of Trypanosoma brucei entry into the brain parenchyma to initiate the second, meningoencephalitic stage of human African trypanosomiasis or sleeping sickness is currently debated and even parasite invasion of the neuropil has been recently questioned. Furthermore, the relationship between neurological features and disease stage are unclear, despite the important diagnostic and therapeutic implications. METHODOLOGY: Using a rat model of chronic Trypanosoma brucei brucei infection we determined the timing of parasite and T-cell neuropil infiltration and its correlation with functional changes. Parasite DNA was detected using trypanosome-specific PCR. Body weight and sleep structure alterations represented by sleep-onset rapid eye movement (SOREM) periods, reported in human and experimental African trypanosomiasis, were monitored. The presence of parasites, as well as CD4+ and CD8+ T-cells in the neuropil was assessed over time in the brain of the same animals by immunocytochemistry and quantitative analyses. PRINCIPAL FINDINGS: Trypanosome DNA was present in the brain at day 6 post-infection and increased more than 15-fold by day 21. Parasites and T-cells were observed in the parenchyma from day 9 onwards. Parasites traversing blood vessel walls were observed in the hypothalamus and other brain regions. Body weight gain was reduced from day 7 onwards. SOREM episodes started in most cases early after infection, with an increase in number and duration after parasite neuroinvasion. CONCLUSION: These findings demonstrate invasion of the neuropil over time, after an initial interval, by parasites and lymphocytes crossing the blood-brain barrier, and show that neurological features can precede this event. The data thus challenge the current clinical and cerebrospinal fluid criteria of disease staging.
Assuntos
Encéfalo/imunologia , Encéfalo/parasitologia , Trypanosoma brucei brucei/imunologia , Tripanossomíase Africana/complicações , Tripanossomíase Africana/imunologia , Animais , Barreira Hematoencefálica , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Doença Crônica , DNA de Helmintos/isolamento & purificação , Modelos Animais de Doenças , Humanos , Neutrófilos/imunologia , Carga Parasitária , Ratos , Sono , Sono REM , Fatores de Tempo , Trypanosoma brucei brucei/genética , Tripanossomíase Africana/parasitologiaRESUMO
In current clinical practice, the initial stage in the detection of pulmonary airway disease is the clinician's interpretation of a bronchoscopy procedure. Due to the nature of this form of detection, diseases are often well advanced upon discovery. This paper proposes a new method for examining the pulmonary airway microcirculatory system via fluorescein imaging techniques similar to those used extensively in ophthalmology. Through the use of a specialized bronchoscopy imaging system we are able to successfully show that fluorescein emission detected in the rabbit trachea exhibits a clear response to inflammatory mediators and thus to changing local vascularity. The clinical implications of this technique are extensive including most importantly the potential use as an evaluation tool for pulmonary disease evaluation and likely other endoscopically accessible organs.
Assuntos
Broncoscopia/métodos , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Mucosa Respiratória/irrigação sanguínea , Mucosa Respiratória/patologia , Vasculite/patologia , Animais , Testes de Provocação Brônquica/métodos , Broncoscópios , Desenho de Equipamento , Análise de Falha de Equipamento , Fluoresceína , Aumento da Imagem/instrumentação , Interpretação de Imagem Assistida por Computador/instrumentação , Mediadores da Inflamação/efeitos adversos , Pulmão/irrigação sanguínea , Pulmão/efeitos dos fármacos , Pulmão/patologia , Microcirculação/efeitos dos fármacos , Microcirculação/patologia , Microscopia de Fluorescência/instrumentação , Coelhos , Mucosa Respiratória/efeitos dos fármacos , Processamento de Sinais Assistido por Computador , Vasculite/induzido quimicamente , Vasculite/complicaçõesRESUMO
PURPOSE: To quantitatively evaluate the rate of cup-to-disc ratio progression in treated patients with primary open-angle glaucoma and to identify clinical factors associated with cup progression. PATIENTS AND METHODS: Fifty one eyes of 51 treated primary open-angle glaucoma patients with a minimum of 9-year longitudinal series of stereoscopic optic disc photographs were studied. Eyes with any other ocular disease except for mild cataract were excluded. Each set of stereoscopic photographs was digitized and viewed stereoscopically on a computer screen using a hand-held stereoscope. Computer-aided planimetry was performed on each set of photographs with examiner-defined cup and disc margins using custom-made software. The software computed linear cup-to-disc ratios as well as peripapillary atrophy area. Both inter-observer and intra-observer reliabilities were evaluated in a masked, random fashion using intra-class correlation. Changes in linear cup-to-disc ratios and peripapillary atrophy were estimated using linear regression over time. All available clinical factors were evaluated for association with the rate of cup progression using a multiple regression model. RESULTS: All patients studied were Caucasian; 31(61%) were females. The mean age at the beginning of the study was 61.6 +/- 7.8 years (range 46-81). The mean follow-up period was 14.3 +/- 3.5 years (median 14.0, range 9.6-22.3). A total of 173 sets of stereo disc photographs were analyzed (3.4 +/- 1.3 per patient). The initial and final linear cup-to-disc ratios were 0.64 +/- 0.15 and 0.73 +/- 0.14 respectively. The inter-rater and intra-rater intraclass correlations were 0.76 (95% CI 0.61-0.87) and 0.97 (95% CI 0.93-0.98) respectively. Using linear regression the rate of linear cup-to-disc ratio change was 0.0068 +/- 0.0062 per year (range -0.0025- 0.0269). Three eyes had an increase in the peripapillary atrophy area. The higher yearly average intraocular pressure was significantly associated with faster rate of linear cup-to-disc ratios progression (P = 0.03). CONCLUSIONS: In treated patients with primary open-angle glaucoma, the rate of progressive optic disc cupping was slow (LCDR progression of 0.0068 per year). The higher yearly average intraocular pressure was significantly associated with a faster rate of cup progression.
Assuntos
Glaucoma de Ângulo Aberto/fisiopatologia , Glaucoma de Ângulo Aberto/terapia , Disco Óptico/fisiopatologia , Doenças do Nervo Óptico/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , Progressão da Doença , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Pressão Intraocular , Masculino , Pessoa de Meia-Idade , FotografaçãoRESUMO
Chronic spinal cord dysfunction occurs in dogs as a consequence of diverse aetiologies, including long-standing spinal cord compression and insidious neurodegenerative conditions. One such neurodegenerative condition is canine degenerative myelopathy (DM), which clinically is a challenge to differentiate from other chronic spinal cord conditions. Although the clinical diagnosis of DM can be strengthened by the identification of the Sod1 mutations that are observed in affected dogs, genetic analysis alone is insufficient to provide a definitive diagnosis. There is a requirement to identify biomarkers that can differentiate conditions with a similar clinical presentation, thus facilitating patient diagnostic and management strategies. A comparison of the cerebrospinal fluid (CSF) protein gel electrophoresis profile between idiopathic epilepsy (IE) and DM identified a protein band that was more prominent in DM. This band was subsequently found to contain a multifunctional protein clusterin (apolipoprotein J) that is protective against endoplasmic reticulum (ER) stress-mediated apoptosis, oxidative stress, and also serves as an extracellular chaperone influencing protein aggregation. Western blot analysis of CSF clusterin confirmed elevated levels in DM compared to IE (p < 0.05). Analysis of spinal cord tissue from DM and control material found that clusterin expression was evident in neurons and that the clusterin mRNA levels from tissue extracts were elevated in DM compared to the control. The plasma clusterin levels was comparable between these groups. However, a comparison of clusterin CSF levels in a number of neurological conditions found that clusterin was elevated in both DM and chronic intervertebral disc disease (cIVDD) but not in meningoencephalitis and IE. These findings indicate that clusterin may potentially serve as a marker for chronic spinal cord disease in the dog; however, additional markers are required to differentiate DM from a concurrent condition such as cIVDD.
Assuntos
Clusterina/líquido cefalorraquidiano , Doenças do Cão/líquido cefalorraquidiano , Doenças da Medula Espinal/veterinária , Animais , Biomarcadores/líquido cefalorraquidiano , Cromatografia Líquida , Doença Crônica , Clusterina/sangue , Clusterina/genética , Doenças do Cão/sangue , Doenças do Cão/patologia , Cães , Eletroforese em Gel de Poliacrilamida , Epilepsia/líquido cefalorraquidiano , Haptoglobinas/líquido cefalorraquidiano , Espectrometria de Massas , Modelos Biológicos , Degeneração Neural/líquido cefalorraquidiano , Degeneração Neural/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologia , Doenças da Medula Espinal/sangue , Doenças da Medula Espinal/líquido cefalorraquidiano , Doenças da Medula Espinal/patologia , Bancos de TecidosRESUMO
Post-herpetic neuralgia (PHN) is the most significant complication of herpes zoster caused by reactivation of latent Varicella-Zoster virus (VZV). We undertook a heterologous infection in vitro study to determine whether PHN-associated VZV isolates induce changes in sodium ion channel currents known to be associated with neuropathic pain. Twenty VZV isolates were studied blind from 11 PHN and 9 non-PHN subjects. Viruses were propagated in the MeWo cell line from which cell-free virus was harvested and applied to the ND7/23-Nav1.8 rat DRG x mouse neuroblastoma hybrid cell line which showed constitutive expression of the exogenous Nav 1.8, and endogenous expression of Nav 1.6 and Nav 1.7 genes all encoding sodium ion channels the dysregulation of which is associated with a range of neuropathic pain syndromes. After 72 hrs all three classes of VZV gene transcripts were detected in the absence of infectious virus. Single cell sodium ion channel recording was performed after 72 hr by voltage-clamping. PHN-associated VZV significantly increased sodium current amplitude in the cell line when compared with non-PHN VZV, wild-type (Dumas) or vaccine VZV strains ((POka, Merck and GSK). These sodium current increases were unaffected by acyclovir pre-treatment but were abolished by exposure to Tetrodotoxin (TTX) which blocks the TTX-sensitive fast Nav 1.6 and Nav 1.7 channels but not the TTX-resistant slow Nav 1.8 channel. PHN-associated VZV sodium current increases were therefore mediated in part by the Nav 1.6 and Nav 1.7 sodium ion channels. An additional observation was a modest increase in message levels of both Nav1.6 and Nav1.7 mRNA but not Nav 1.8 in PHN virally infected cells.
Assuntos
Herpesvirus Humano 3/genética , Canal de Sódio Disparado por Voltagem NAV1.8/genética , Neuralgia Pós-Herpética/genética , Aciclovir/farmacologia , Animais , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Herpes Zoster/complicações , Herpes Zoster/genética , Herpes Zoster/virologia , Herpesvirus Humano 3/patogenicidade , Humanos , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Neuralgia Pós-Herpética/etiologia , Neuralgia Pós-Herpética/patologia , Neuralgia Pós-Herpética/virologia , Ratos , Tetrodotoxina/farmacologiaRESUMO
Reduced cardiovascular fitness (CVF) is a risk factor for obesity and cardiovascular disease. It has previously shown that a school-based fitness curriculum can improve CVF, and other health indicators in middle school aged children. Whether an afterschool program improves CVF and other health markers in elementary-school children is unresolved. The objective of this study was therefore to determine whether an on-site afterschool-based fitness program improves body composition, cardiovascular fitness level, in elementary school children. 80 elementary school children were evaluated in a "fitness-oriented" afterschool program managed by the local YMCA. Children underwent evaluation of cardiovascular fitness by maximal VO2 treadmill testing and body composition by dual x-ray absorptiometry (DXA), at baseline (prior to the school-year) and again at end of the school year. Findings revealed that, at baseline, children had a mean age of 8.8 years, BMI of 18.7± 3, with a maximal VO2 of 40.03 ± 7.6 ml/kg/min, and percent body fat of 28.7 ± 7%. After a 9-month intervention, children maximal VO2 increased to 44.8 ± 7.5 ml/kg/min (p=0.04) and percent body fat decreased to 25.8 ± 6.2% (p=0.033). The study concluded that on-site afterschool programming focusing on fitness improved body composition and cardiovascular fitness, in elementary school children. Combined with prior studies, these data demonstrate that afterschool-based fitness curricula can benefit both obese and non-obese children. It was therefore recommended that, partnerships with schools to promote fitness even outside of school time should be a part of a school approach to improving children's health.
RESUMO
We demonstrate that intravenous delivery of human, or rat, pancreas-derived pathfinder (PDP) cells can totally regenerate critically damaged adult tissue and restore normal function across a species barrier. We have used a mouse model of streptozotocin (STZ)-induced diabetes to demonstrate this. Normoglycemia was restored and maintained for up to 89 days following the induction of diabetes and subsequent intravenous delivery of PDP cells. Normal pancreatic histology also appeared to be restored, and treated diabetic animals gained body weight. Regenerated tissue was primarily of host origin, with few rat or human cells detectable by fluorescent in situ hybridization (FISH). Crucially, the insulin produced by these animals was overwhelmingly murine in origin and was both types I and II, indicative of a process of developmental recapitulation. These results demonstrate the feasibility of using intravenous administration of adult cells to regenerate damaged tissue. Critically, they enhance our understanding of the mechanisms relating to such repair and suggest a means for novel therapeutic intervention in loss of tissue and organ function with age.
Assuntos
Diabetes Mellitus Experimental/terapia , Pâncreas/citologia , Pâncreas/fisiologia , Regeneração , Transplante de Células-Tronco , Adulto , Animais , Diabetes Mellitus Experimental/patologia , Feminino , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas/patologia , RatosRESUMO
It is widely thought that demyelination contributes to the degeneration of axons and, in combination with acute inflammatory injury, is responsible for progressive axonal loss and persistent clinical disability in inflammatory demyelinating disease. In this study we sought to characterize the relationship between demyelination, inflammation and axonal transport changes using a Plp1-transgenic mouse model of Pelizaeus-Merzbacher disease. In the optic pathway of this non-immune mediated model of demyelination, myelin loss progresses from the optic nerve head towards the brain, over a period of months. Axonal transport is functionally perturbed at sites associated with local inflammation and 'damaged' myelin. Surprisingly, where demyelination is complete, naked axons appear well preserved despite a significant reduction of axonal transport. Our results suggest that neuroinflammation and/or oligodendrocyte dysfunction are more deleterious for axonal health than demyelination per se, at least in the short term.