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BACKGROUND: Type I interferons (IFN-I) are fundamental in controlling viral infections but fatal interferonopathy is restricted in the immune-privileged central nervous system (CNS). In contrast to the well-established role of Interferon Regulatory Factor 7 (IRF7) in the regulation of IFN-I response in the periphery, little is known about the specific function in the CNS. METHODS: To investigate the role for IRF7 in antiviral response during neurotropic virus infection, mice deficient for IRF3 and IRF7 were infected systemically with Langat virus (LGTV). Viral burden and IFN-I response was analyzed in the periphery and the CNS by focus formation assay, RT-PCR, immunohistochemistry and in vivo imaging. Microglia and infiltration of CNS-infiltration of immune cells were characterized by flow cytometry. RESULTS: Here, we demonstrate that during infection with the neurotropic Langat virus (LGTV), an attenuated member of the tick-borne encephalitis virus (TBEV) subgroup, neurons do not rely on IRF7 for cell-intrinsic antiviral resistance and IFN-I induction. An increased viral replication in IRF7-deficient mice suggests an indirect antiviral mechanism. Astrocytes rely on IRF7 to establish a cell-autonomous antiviral response. Notably, the loss of IRF7 particularly in astrocytes resulted in a high IFN-I production. Sustained production of IFN-I in astrocytes is independent of an IRF7-mediated positive feedback loop. CONCLUSION: IFN-I induction in the CNS is profoundly regulated in a cell type-specific fashion.
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Encefalitis Transmitida por Garrapatas , Factor 7 Regulador del Interferón , Interferón Tipo I , Animales , Ratones , Anticuerpos , Astrocitos , Sistema Nervioso Central , Factor 7 Regulador del Interferón/genética , Encefalitis Transmitida por Garrapatas/inmunologíaRESUMEN
Microtubules (MTs) are central components of the neuronal cytoskeleton and play a critical role in CNS integrity, function, and plasticity. Neuronal MTs are diverse due to extensive post-translational modifications (PTMs), particularly detyrosination/tyrosination, in which the C-terminal tyrosine of α-tubulin is cyclically removed by a carboxypeptidase and reattached by a tubulin-tyrosine ligase (TTL). The detyrosination/tyrosination cycle of MTs has been shown to be an important regulator of MT dynamics in neurons. TTL-null mice exhibit impaired neuronal organization and die immediately after birth, indicating TTL function is vital to the CNS. However, the detailed cellular role of TTL during development and in the adult brain remains elusive. Here, we demonstrate that conditional deletion of TTL in the neocortex and hippocampus during network development results in a pathophysiological phenotype defined by incomplete development of the corpus callosum and anterior commissures due to axonal growth arrest. TTL loss was also associated with a deficit in spatial learning, impaired synaptic plasticity, and reduced number of spines in hippocampal neurons, suggesting that TTL also plays a critical role in hippocampal network development. TTL deletion after postnatal development, specifically in the hippocampus and in cultured hippocampal neurons, led to a loss of spines and impaired spine structural plasticity. This indicates a novel and important function of TTL for synaptic plasticity in the adult brain. In conclusion, this study reveals the importance of α-tubulin tyrosination, which defines the dynamics of MTs, in controlling proper network formation and suggests TTL-mediated tyrosination as a new key determinant of synaptic plasticity in the adult brain.
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Chronic antibody-mediated rejection (AMR) is a key limiting factor for the clinical outcome of a kidney transplantation (Ktx), where early diagnosis and therapeutic intervention is needed. This study describes the identification of the biomarker CXC-motif chemokine ligand (CXCL) 9 as an indicator for AMR and presents a new aptamer-antibody-hybrid lateral flow assay (hybrid-LFA) for detection in urine. Biomarker evaluation included two independent cohorts of kidney transplant recipients (KTRs) from a protocol biopsy program and used subgroup comparisons according to BANFF-classifications. Plasma, urine and biopsy lysate samples were analyzed with a Luminex-based multiplex assay. The CXCL9-specific hybrid-LFA was developed based upon a specific rat antibody immobilized on a nitrocellulose-membrane and the coupling of a CXCL9-binding aptamer to gold nanoparticles. LFA performance was assessed according to receiver operating characteristic (ROC) analysis. Among 15 high-scored biomarkers according to a neural network analysis, significantly higher levels of CXCL9 were found in plasma and urine and biopsy lysates of KTRs with biopsy-proven AMR. The newly developed hybrid-LFA reached a sensitivity and specificity of 71% and an AUC of 0.79 for CXCL9. This point-of-care-test (POCT) improves early diagnosis-making in AMR after Ktx, especially in KTRs with undetermined status of donor-specific HLA-antibodies.
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Influenza A virus (IAV) causes respiratory tract disease and is responsible for seasonal and reoccurring epidemics affecting all age groups. Next to typical disease symptoms, such as fever and fatigue, IAV infection has been associated with behavioral alterations presumably contributing to the development of major depression. Previous experiments using IAV/H1N1 infection models have shown impaired hippocampal neuronal morphology and cognitive abilities, but the underlying pathways have not been fully described. In this study, we demonstrate that infection with a low-dose non-neurotrophic H1N1 strain of IAV causes ample peripheral immune response followed by a temporary blood-brain barrier disturbance. Although histological examination did not reveal obvious pathological processes in the brains of IAV-infected mice, detailed multidimensional flow cytometric characterization of immune cells uncovered subtle alterations in the activation status of microglial cells. More specifically, we detected an altered expression pattern of major histocompatibility complex classes I and II, CD80, and F4/80 accompanied by elevated mRNA levels of CD36, CD68, C1QA, and C3, suggesting evolved synaptic pruning. To closer evaluate how these profound changes affect synaptic balance, we established a highly sensitive multiplex flow cytometry-based approach called flow synaptometry. The introduction of this novel technique enabled us to simultaneously quantify the abundance of pre- and postsynapses from distinct brain regions. Our data reveal a significant reduction of VGLUT1 in excitatory presynaptic terminals in the cortex and hippocampus, identifying a subtle dysbalance in glutamatergic synapse transmission upon H1N1 infection in mice. In conclusion, our results highlight the consequences of systemic IAV-triggered inflammation on the central nervous system and the induction and progression of neuronal alterations. IMPORTANCE Influenza A virus (IAV) causes mainly respiratory tract disease with fever and fatigue but is also associated with behavioral alterations in humans. Here, we demonstrate that infection with a low-dose non-neurotrophic H1N1 strain of IAV causes peripheral immune response followed by a temporary blood-brain barrier disturbance. Characterization of immune cells uncovered subtle alterations in the activation status of microglia cells that might reshape neuronal synapses. We established a highly sensitive multiplex flow cytometry-based approach called flow synaptometry to more closely study the synapses. Thus, we detected a specific dysbalance in glutamatergic synapse transmission upon H1N1 infection in mice. In conclusion, our results highlight the consequences of systemic IAV-triggered inflammation on the central nervous system and the induction and progression of neuronal alterations.
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Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Virus de la Influenza A , Microglía/metabolismo , Transmisión Sináptica/fisiología , Animales , Encéfalo/patología , Quimiocinas , Citocinas , Expresión Génica , Humanos , Inflamación/virología , Virus de la Influenza A/genética , Gripe Humana/virología , Ratones , Infecciones por Orthomyxoviridae/virologíaRESUMEN
Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation. Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (αDEC-205) for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting αDEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to αDEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA). We have successfully used chemically crosslinked αDEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.
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Adyuvantes Inmunológicos/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Antígenos CD/inmunología , Células Dendríticas/inmunología , Inmunidad Celular/inmunología , Lectinas Tipo C/inmunología , Antígenos de Histocompatibilidad Menor/inmunología , Receptores de Superficie Celular/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Antígenos CD/metabolismo , Reactividad Cruzada , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Femenino , Técnicas In Vitro , Lectinas Tipo C/metabolismo , Ratones , Ratones Endogámicos C57BL , Antígenos de Histocompatibilidad Menor/metabolismo , Ovalbúmina/inmunología , Receptores de Superficie Celular/metabolismoRESUMEN
Self-propagating amyloid-ß (Aß) aggregates or seeds possibly drive pathogenesis of Alzheimer's disease (AD). Small molecules targeting such structures might act therapeutically in vivo. Here, a fluorescence polarization assay was established that enables the detection of compound effects on both seeded and spontaneous Aß42 aggregation. In a focused screen of anti-amyloid compounds, we identified Disperse Orange 1 (DO1) ([4-((4-nitrophenyl)diazenyl)-N-phenylaniline]), a small molecule that potently delays both seeded and non-seeded Aß42 polymerization at substoichiometric concentrations. Mechanistic studies revealed that DO1 disrupts preformed fibrillar assemblies of synthetic Aß42 peptides and decreases the seeding activity of Aß aggregates from brain extracts of AD transgenic mice. DO1 also reduced the size and abundance of diffuse Aß plaques and decreased neuroinflammation-related gene expression changes in brains of 5xFAD transgenic mice. Finally, improved nesting behavior was observed upon treatment with the compound. Together, our evidence supports targeting of self-propagating Aß structures with small molecules as a valid therapeutic strategy.
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Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/antagonistas & inhibidores , Compuestos Azo/farmacología , Colorantes/farmacología , Inflamación/tratamiento farmacológico , Placa Amiloide/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Compuestos Azo/química , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Colorantes/química , Relación Dosis-Respuesta a Droga , Femenino , Inflamación/genética , Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Estructura Molecular , Placa Amiloide/genética , Placa Amiloide/metabolismo , Polimerizacion/efectos de los fármacos , Agregado de Proteínas/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Type II diabetes (T2D) is characterized by diminished insulin production and resistance of cells to insulin. Among others, endoplasmic reticulum (ER) stress is a principal factor contributing to T2D and induces a shift towards a more reducing cellular environment. At the same time, peripheral insulin resistance triggers the over-production of regulatory hormones such as insulin and human islet amyloid polypeptide (hIAPP). We show that the differential aggregation of reduced and oxidized hIAPP assists to maintain the redox equilibrium by restoring redox equivalents. Aggregation thus induces redox balancing which can assist initially to counteract ER stress. Failure of the protein degradation machinery might finally result in ß-cell disruption and cell death. We further present a structural characterization of hIAPP in solution, demonstrating that the N-terminus of the oxidized peptide has a high propensity to form an α-helical structure which is lacking in the reduced state of hIAPP. In healthy cells, this residual structure prevents the conversion into amyloidogenic aggregates.
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Diabetes Mellitus Tipo 2/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/química , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Animales , Diabetes Mellitus Tipo 2/patología , Estrés del Retículo Endoplásmico , Femenino , Humanos , Ratones Endogámicos BALB C , Ratones Transgénicos , Oxidación-Reducción , Agregación Patológica de Proteínas , Conformación ProteicaRESUMEN
Adapters bind motor proteins to cargoes and therefore play essential roles in Kinesin-1 mediated intracellular transport. The regulatory mechanisms governing adapter functions and the spectrum of cargoes recognized by individual adapters remain poorly defined. Here, we show that cargoes transported by the Kinesin-1 adapter FEZ1 are enriched for presynaptic components and identify that specific phosphorylation of FEZ1 at its serine 58 regulatory site is mediated by microtubule affinity-regulating kinases (MARK/PAR-1). Loss of MARK/PAR-1 impairs axonal transport, with adapter and cargo abnormally co-aggregating in neuronal cell bodies and axons. Presynaptic specializations are markedly reduced and distorted in FEZ1 and MARK/PAR-1 mutants. Strikingly, abnormal co-aggregates of unphosphorylated FEZ1, Kinesin-1 and its putative cargoes are present in brains of transgenic mice modelling aspects of Alzheimer's disease, a neurodegenerative disorder exhibiting impaired axonal transport and altered MARK activity. Our findings suggest that perturbed FEZ1-mediated synaptic delivery of proteins arising from abnormal signalling potentially contributes to the process of neurodegeneration.
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Transporte Axonal/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas Serina-Treonina Quinasas/genética , Vesículas Sinápticas/metabolismo , Proteínas Supresoras de Tumor/genética , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Cinesinas/genética , Ratones , Mutación , Neuronas/metabolismo , Neuronas/patología , Fosforilación , Proteínas Serina-Treonina Quinasas/deficiencia , Ratas , Transmisión Sináptica , Vesículas Sinápticas/patología , Proteínas Supresoras de Tumor/deficienciaRESUMEN
Erythropoietin (EPO) exerts potent neuroprotective, neuroregenerative and procognitive functions. However, unequivocal demonstration of erythropoietin receptor (EPOR) expression in brain cells has remained difficult since previously available anti-EPOR antibodies (EPOR-AB) were unspecific. We report here a new, highly specific, polyclonal rabbit EPOR-AB directed against different epitopes in the cytoplasmic tail of human and murine EPOR and its characterization by mass spectrometric analysis of immuno-precipitated endogenous EPOR, Western blotting, immunostaining and flow cytometry. Among others, we applied genetic strategies including overexpression, Lentivirus-mediated conditional knockout of EpoR and tagged proteins, both on cultured cells and tissue sections, as well as intracortical implantation of EPOR-transduced cells to verify specificity. We show examples of EPOR expression in neurons, oligodendroglia, astrocytes and microglia. Employing this new EPOR-AB with double-labeling strategies, we demonstrate membrane expression of EPOR as well as its localization in intracellular compartments such as the Golgi apparatus. Moreover, we show injury-induced expression of EPOR. In mice, a stereotactically applied stab wound to the motor cortex leads to distinct EpoR expression by reactive GFAP-expressing cells in the lesion vicinity. In a patient suffering from epilepsy, neurons and oligodendrocytes of the hippocampus strongly express EPOR. To conclude, this new analytical tool will allow neuroscientists to pinpoint EPOR expression in cells of the nervous system and to better understand its role in healthy conditions, including brain development, as well as under pathological circumstances, such as upregulation upon distress and injury.
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Small GTPases of the Rab family not only regulate target recognition in membrane traffic but also control other cellular functions such as cytoskeletal transport and autophagy. Here we show that Rab26 is specifically associated with clusters of synaptic vesicles in neurites. Overexpression of active but not of GDP-preferring Rab26 enhances vesicle clustering, which is particularly conspicuous for the EGFP-tagged variant, resulting in a massive accumulation of synaptic vesicles in neuronal somata without altering the distribution of other organelles. Both endogenous and induced clusters co-localize with autophagy-related proteins such as Atg16L1, LC3B and Rab33B but not with other organelles. Furthermore, Atg16L1 appears to be a direct effector of Rab26 and binds Rab26 in its GTP-bound form, albeit only with low affinity. We propose that Rab26 selectively directs synaptic and secretory vesicles into preautophagosomal structures, suggesting the presence of a novel pathway for degradation of synaptic vesicles.
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Autofagia , Vesículas Sinápticas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Cuerpo Celular/metabolismo , Compartimento Celular , Células Cultivadas , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Hipocampo/citología , Humanos , Ratones Endogámicos BALB C , Proteínas Mutantes/metabolismo , Unión Neuromuscular/metabolismo , Unión Neuromuscular/ultraestructura , Neuronas/citología , Neuronas/metabolismo , Neuronas/ultraestructura , Fagosomas/metabolismo , Ratas , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Inhibitory (GABAergic) interneurons entrain assemblies of excitatory principal neurons to orchestrate information processing in the hippocampus. Disrupting the dynamic recruitment as well as the temporally precise activity of interneurons in hippocampal circuitries can manifest in epileptiform seizures, and impact specific behavioral traits. Despite the importance of GABAergic interneurons during information encoding in the brain, experimental tools to selectively manipulate GABAergic neurotransmission are limited. Here, we report the selective elimination of GABAergic interneurons by a ribosome inactivation approach through delivery of saporin-conjugated anti-vesicular GABA transporter antibodies (SAVAs) in vitro as well as in the mouse and rat hippocampus in vivo. We demonstrate the selective loss of GABAergic--but not glutamatergic--synapses, reduced GABA release, and a shift in excitation/inhibition balance in mixed cultures of hippocampal neurons exposed to SAVAs. We also show the focal and indiscriminate loss of calbindin(+), calretinin(+), parvalbumin/system A transporter 1(+), somatostatin(+), vesicular glutamate transporter 3 (VGLUT3)/cholecystokinin/CB(1) cannabinoid receptor(+) and neuropeptide Y(+) local-circuit interneurons upon SAVA microlesions to the CA1 subfield of the rodent hippocampus, with interneuron debris phagocytosed by infiltrating microglia. SAVA microlesions did not affect VGLUT1(+) excitatory afferents. Yet SAVA-induced rearrangement of the hippocampal circuitry triggered network hyperexcitability associated with the progressive loss of CA1 pyramidal cells and the dispersion of dentate granule cells. Overall, our data identify SAVAs as an effective tool to eliminate GABAergic neurons from neuronal circuits underpinning high-order behaviors and cognition, and whose manipulation can recapitulate pathogenic cascades of epilepsy and other neuropsychiatric illnesses.
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Neuronas GABAérgicas/fisiología , Hipocampo , Interneuronas , Red Nerviosa/fisiología , Inhibición Neural , Fagocitosis , Animales , Calbindina 2 , Calbindinas , Muerte Celular/genética , Células Cultivadas , Femenino , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/patología , Hipocampo/metabolismo , Hipocampo/patología , Hipocampo/fisiología , Interneuronas/metabolismo , Interneuronas/patología , Interneuronas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Red Nerviosa/metabolismo , Red Nerviosa/patología , Inhibición Neural/genética , Fagocitosis/genética , Conejos , Ratas , Ratas Sprague-Dawley , Proteína G de Unión al Calcio S100/genéticaRESUMEN
Staphylococcus aureus is a major human pathogen. Superantigens (SAg) are important virulence factors in S. aureus, but the regulation of SAg gene expression is largely unknown. Using 2 sequenced S. aureus strains (COL and Newman) and 4 clinical isolates, regulation of gene expression was investigated in more detail for 12 SAgs. The SAg-encoding genes were expressed in a growth phase-dependent manner: while the egc operon was mainly transcribed at low optical densities, the transcription of seb was induced at high optical densities. The transcript levels of sea, sek, seq, sep, and tst-1 did not change significantly during growth. The T cell-mitogenic activity of supernatants correlated with the transcription data. SaeRS and σ(B) strongly influenced SAg gene transcription. σ(B) activated transcription of seh, tst-1, and of the egc operon. A possible σ(B)-dependent promoter was identified in front of the egc operon. In contrast, a loss of σ(B) enhanced the transcript level of seb, suggesting an indirect effect of the alternative sigma factor on the transcription of this gene. Transcriptional studies of an saeS mutant showed that the two-component system only activates transcription of seb. The influence of σ(B) and SaeRS on the expression of SAg genes was validated by T cell proliferation assays. For sigB mutants in different strains, different effects on the T cell-mitogenic potential were observed depending on the SAg gene repertoire of the isolates.
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Proteínas Bacterianas/metabolismo , Enterotoxinas/genética , Factor sigma/metabolismo , Staphylococcus aureus/genética , Superantígenos/metabolismo , Proteínas Bacterianas/genética , Proliferación Celular , Células Cultivadas , Enterotoxinas/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Humanos , Mutación , Operón , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Proteínas Quinasas , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Factor sigma/genética , Staphylococcus aureus/clasificación , Staphylococcus aureus/aislamiento & purificación , Superantígenos/genética , Factores de Transcripción , Transcripción Genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Staphylococcus aureus is the most common cause of nosocomial infections. Multiple antibiotic resistance and severe clinical outcomes provide a strong rationale for development of immunoglobulin-based strategies. Traditionally, novel immunological approaches against bacterial pathogens involve antibodies directed against cell surface-exposed virulence-associated epitopes or toxins. In this study, we generated a monoclonal antibody targeting the housekeeping protein IsaA, a suggested soluble lytic transglycosylase of S. aureus, and tested its therapeutic efficacy in two experimental mouse infection models. A murine anti-IsaA antibody of the IgG1 subclass (UK-66P) showed the highest binding affinity in Biacore analysis. This antibody recognized all S. aureus strains tested, including hospital-acquired and community-acquired methicillin-resistant S. aureus strains. Therapeutic efficacy in vivo in mice was analyzed using a central venous catheter-related infection model and a sepsis survival model. In both models, anti-IsaA IgG1 conferred protection against staphylococcal infection. Ex vivo, UK-66P activates professional phagocytes and induces highly microbicidal reactive oxygen metabolites in a dose-dependent manner, resulting in bacterial killing. The study provides proof of concept that monoclonal IgG1 antibodies with high affinity to the ubiquitously expressed, single-epitope-targeting IsaA are effective in the treatment of staphylococcal infection in different mouse models. Anti-IsaA antibodies might be a useful component in an antibody-based therapeutic for prophylaxis or adjunctive treatment of human cases of S. aureus infections.
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Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/uso terapéutico , Antígenos Bacterianos/inmunología , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Animales , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Masculino , Ratones , Sepsis/tratamiento farmacológico , Sepsis/microbiología , Infecciones Estafilocócicas/microbiologíaRESUMEN
BACKGROUND: Methylation of residues in histone tails is part of a network that regulates gene expression. JmjC domain containing proteins catalyze the oxidative removal of methyl groups on histone lysine residues. Here, we report studies to test the involvement of Jumonji domain-containing protein 6 (Jmjd6) in histone lysine demethylation. Jmjd6 has recently been shown to hydroxylate RNA splicing factors and is known to be essential for the differentiation of multiple tissues and cells during embryogenesis. However, there have been conflicting reports as to whether Jmjd6 is a histone-modifying enzyme. METHODOLOGY/PRINCIPAL FINDINGS: Immunolocalization studies reveal that Jmjd6 is distributed throughout the nucleoplasm outside of regions containing heterochromatic DNA, with occasional localization in nucleoli. During mitosis, Jmjd6 is excluded from the nucleus and reappears in the telophase of the cell cycle. Western blot analyses confirmed that Jmjd6 forms homo-multimers of different molecular weights in the nucleus and cytoplasm. A comparison of mono-, di-, and tri-methylation states of H3K4, H3K9, H3K27, H3K36, and H4K20 histone residues in wildtype and Jmjd6-knockout cells indicate that Jmjd6 is not involved in the demethylation of these histone lysine residues. This is further supported by overexpression of enzymatically active and inactive forms of Jmjd6 and subsequent analysis of histone methylation patterns by immunocytochemistry and western blot analysis. Finally, treatment of cells with RNase A and DNase I indicate that Jmjd6 may preferentially associate with RNA/RNA complexes and less likely with chromatin. CONCLUSIONS/SIGNIFICANCE: Taken together, our results provide further evidence that Jmjd6 is unlikely to be involved in histone lysine demethylation. We confirmed that Jmjd6 forms multimers and showed that nuclear localization of the protein involves association with a nucleic acid matrix.
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Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Western Blotting , Núcleo Celular/metabolismo , Técnica del Anticuerpo Fluorescente , Eliminación de Gen , Técnicas de Inactivación de Genes , Histona Demetilasas con Dominio de Jumonji/genética , MetilaciónRESUMEN
N-terminally truncated Aß peptides starting with pyroglutamate (AßpE3) represent a major fraction of all Aß peptides in the brain of Alzheimer disease (AD) patients. AßpE3 has a higher aggregation propensity and stability and shows increased toxicity compared with full-length Aß. In the present work, we generated a novel monoclonal antibody (9D5) that selectively recognizes oligomeric assemblies of AßpE3 and studied the potential involvement of oligomeric AßpE3 in vivo using transgenic mouse models as well as human brains from sporadic and familial AD cases. 9D5 showed an unusual staining pattern with almost nondetectable plaques in sporadic AD patients and non-demented controls. Interestingly, in sporadic and familial AD cases prominent intraneuronal and blood vessel staining was observed. Using a novel sandwich ELISA significantly decreased levels of oligomers in plasma samples from patients with AD compared with healthy controls were identified. Moreover, passive immunization of 5XFAD mice with 9D5 significantly reduced overall Aß plaque load and AßpE3 levels, and normalized behavioral deficits. These data indicate that 9D5 is a therapeutically and diagnostically effective monoclonal antibody targeting low molecular weight AßpE3 oligomers.
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Enfermedad de Alzheimer/metabolismo , Ácido Pirrolidona Carboxílico/química , Amiloide/química , Animales , Conducta Animal , Encéfalo/metabolismo , Línea Celular Tumoral , Cromatografía/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Inmunoquímica/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Peso Molecular , Estructura Terciaria de Proteína , TransgenesRESUMEN
Side-chain oligo- and polyglutamylation represents an important posttranslational modification in tubulin physiology. The particular number of glutamate units is related to specific regulatory functions. In this work, we present a method for the synthesis of building blocks for the Fmoc synthesis of peptides containing main chain glutamic acid residues that carry side-chain branching with oligo-glutamic acid. The two model peptide sequences CYEEVGVDSVEGEG-E(E(x))-EEGEEY and CQDATADEQG-E(E(x))-FEEEEGEDEA from the C-termini of mammalian α1- and ß1-tubulin, respectively, containing oligo-glutamic acid side-chain branching with lengths of 1 to 5 amino acids were assembled in good yield and purity. The products may lead to the generation of specific antibodies which should be important tools for a more detailed investigation of polyglutamylation processes.
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A new approach for the detection of virulence factors of Staphylococcus aureus and Staphylococcus epidermidis using an electrical protein array chip technology is presented. The procedure is based on an enzyme-linked sandwich immunoassay, which includes recognition and binding of virulence factors by specific capture and detection antibodies. Detection of antibody-bound virulence factors is achieved by measuring the electrical current generated by redox recycling of an enzymatically released substance. The current (measured in nanoampere) corresponds to the amount of the target molecule in the analyzed sample. The electrical protein chip allows for a fast detection of Staphylococcus enterotoxin B (SEB) of S. aureus and immunodominant antigen A homologue (IsaA homologue) of S. epidermidis in different liquid matrices. The S. aureus SEB virulence factor could be detected in minimal medium, milk, and urine in a concentration of 1 ng/ml within less than 23 min. Furthermore, a simultaneous detection of SEB of S. aureus and IsaA homologue of S. epidermidis in a single assay could be demonstrated.
Asunto(s)
Antígenos Bacterianos/análisis , Enterotoxinas/análisis , Análisis por Matrices de Proteínas , Staphylococcus aureus/química , Staphylococcus epidermidis/inmunología , Factores de Virulencia/análisis , Animales , Anticuerpos Antibacterianos/análisis , Anticuerpos Antibacterianos/inmunología , Anticuerpos Inmovilizados , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Técnicas Biosensibles , Electroquímica/métodos , Enterotoxinas/inmunología , Enterotoxinas/orina , Contaminación de Alimentos , Microbiología de Alimentos , Leche/química , Factores de Virulencia/inmunología , Factores de Virulencia/orinaRESUMEN
Neurotransmitter uptake into synaptic vesicles is mediated by vesicular neurotransmitter transporters. Although these transporters belong to different families, they all are thought to share a common overall topology with an even number of transmembrane domains. Using epitope-specific antibodies and mass spectrometry we show that the vesicular GABA transporter (VGAT) possesses an uneven number of transmembrane domains, with the N terminus facing the cytoplasm and the C terminus residing in the synaptic vesicle lumen. Antibodies recognizing the C terminus of VGAT (anti-VGAT-C) selectively label GABAergic nerve terminals of live cultured hippocampal and striatal neurons as confirmed by immunocytochemistry and patch-clamp electrophysiology. Injection of fluorochromated anti-VGAT-C into the hippocampus of mice results in specific labeling of GABAergic synapses in vivo. Overall, our data open the possibility of studying novel GABA release sites, characterizing inhibitory vesicle trafficking, and establishing their contribution to inhibitory neurotransmission at identified GABAergic synapses.
Asunto(s)
Inmunohistoquímica/métodos , Prosencéfalo/metabolismo , Coloración y Etiquetado/métodos , Sinapsis/metabolismo , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/química , Ácido gamma-Aminobutírico/metabolismo , Animales , Especificidad de Anticuerpos , Cuerpo Estriado/metabolismo , Cuerpo Estriado/ultraestructura , Endocitosis/fisiología , Exocitosis/fisiología , Hipocampo/metabolismo , Hipocampo/ultraestructura , Espectrometría de Masas , Ratones , Inhibición Neural/fisiología , Técnicas de Placa-Clamp , Prosencéfalo/ultraestructura , Estructura Terciaria de Proteína/fisiología , Sinapsis/ultraestructura , Membranas Sinápticas/metabolismo , Membranas Sinápticas/ultraestructura , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestructura , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/inmunología , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismoRESUMEN
Cyclic-diguanylate (c-di-GMP) is a widespread bacterial signal molecule that plays a major role in the modulation of cellular surface components, such as exopolysaccharides and fimbriae, and in the establishment of a sessile life style. Here, we report that intracellular c-di-GMP levels influence cupA-encoded fimbriae expression in Pseudomonas aeruginosa. In an autoaggregative P. aeruginosa small colony variant (SCV) CupA fimbriae and the intracellular c-di-GMP concentration were found to be enhanced as compared with the clonal wild-type. The SCV morphology and the expression of CupA fimbriae were dependent on a functional PA1120 and morA gene both encoding a GGDEF domain. Overexpression of the GGDEF domain protein PA1120 complemented the PA1120 and the morA mutant with respect to CupA fimbriae expression. In agreement with these findings, overexpression of the EAL domain containing phenotypic variance regulator (PvrR) in the SCV resulted in a decreased intracellular level of c-di-GMP, a reduced cupA fimbriae expression and a switch to wild-type colony morphology.
Asunto(s)
GMP Cíclico/análogos & derivados , Proteínas Fimbrias/biosíntesis , Fimbrias Bacterianas/metabolismo , Pseudomonas aeruginosa/metabolismo , GMP Cíclico/metabolismo , Proteínas Fimbrias/genética , Fimbrias Bacterianas/genética , Humanos , Fenotipo , Estructura Terciaria de Proteína , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/aislamiento & purificaciónRESUMEN
Tubulin is subject to a special cycle of detyrosination/tyrosination in which the C-terminal tyrosine of alpha-tubulin is cyclically removed by a carboxypeptidase and readded by a tubulin-tyrosine-ligase (TTL). This tyrosination cycle is conserved in evolution, yet its physiological importance is unknown. Here, we find that TTL suppression in mice causes perinatal death. A minor pool of tyrosinated (Tyr-)tubulin persists in TTL null tissues, being present mainly in dividing TTL null cells where it originates from tubulin synthesis, but it is lacking in postmitotic TTL null cells such as neurons, which is apparently deleterious because early death in TTL null mice is, at least in part, accounted for by a disorganization of neuronal networks, including a disruption of the cortico-thalamic loop. Correlatively, cultured TTL null neurons display morphogenetic anomalies including an accelerated and erratic time course of neurite outgrowth and a premature axonal differentiation. These anomalies may involve a mislocalization of CLIP170, which we find lacking in neurite extensions and growth cones of TTL null neurons. Our results demonstrate a vital role of TTL for neuronal organization and suggest a requirement of Tyr-tubulin for proper control of neurite extensions.