RESUMO
While a variety of therapeutic options continue to emerge for COVID-19 treatment, convalescent plasma (CP) has been used as a possible treatment option early in the pandemic. One of the most significant challenges with CP therapy, however, both when defining its efficacy and implementing its approach clinically, is accurately and efficiently characterizing an otherwise heterogenous therapeutic treatment. Given current limitations, our goal is to leverage a SARS antibody testing platform with a newly developed automated endpoint titer analysis program to rapidly define SARS-CoV-2 antibody levels in CP donors and hospitalized patients. A newly developed antibody detection platform was used to perform a serial dilution enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (Ig)G, IgM, and IgA SARS-CoV-2 antibodies. Data were then analyzed using commercially available software, GraphPad Prism, or a newly developed program developed in Python called TiterScape, to analyze endpoint titers. Endpoint titer calculations and analysis times were then compared between the two analysis approaches. Serial dilution analysis of SARS-CoV-2 antibody levels revealed a high level of heterogeneity between individuals. Commercial platform analysis required significant time for manual data input and extrapolated endpoint titer values when the last serial dilution was above the endpoint cutoff, occasionally producing erroneously high results. By contrast, TiterScape processed 1008 samples for endpoint titer results in roughly 14 minutes compared with the 8 hours required for the commercial software program analysis. Equally important, results generated by TiterScape and Prism were highly similar, with differences averaging 1.26 ± 0.2 percent (mean ± SD). The pandemic has created unprecedented challenges when seeking to accurately test large numbers of individuals for SARS-CoV-2 antibody levels with a rapid turnaround time. ELISA platforms capable of serial dilution analysis coupled with a highly flexible software interface may provide a useful tool when seeking to define endpoint titers in a high-throughput manner. Immunohematology 2021;37:33-43.While a variety of therapeutic options continue to emerge for COVID-19 treatment, convalescent plasma (CP) has been used as a possible treatment option early in the pandemic. One of the most significant challenges with CP therapy, however, both when defining its efficacy and implementing its approach clinically, is accurately and efficiently characterizing an otherwise heterogenous therapeutic treatment. Given current limitations, our goal is to leverage a SARS antibody testing platform with a newly developed automated endpoint titer analysis program to rapidly define SARS-CoV-2 antibody levels in CP donors and hospitalized patients. A newly developed antibody detection platform was used to perform a serial dilution enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (Ig)G, IgM, and IgA SARS-CoV-2 antibodies. Data were then analyzed using commercially available software, GraphPad Prism, or a newly developed program developed in Python called TiterScape, to analyze endpoint titers. Endpoint titer calculations and analysis times were then compared between the two analysis approaches. Serial dilution analysis of SARS-CoV-2 antibody levels revealed a high level of heterogeneity between individuals. Commercial platform analysis required significant time for manual data input and extrapolated endpoint titer values when the last serial dilution was above the endpoint cutoff, occasionally producing erroneously high results. By contrast, TiterScape processed 1008 samples for endpoint titer results in roughly 14 minutes compared with the 8 hours required for the commercial software program analysis. Equally important, results generated by TiterScape and Prism were highly similar, with differences averaging 1.26 ± 0.2 percent (mean ± SD). The pandemic has created unprecedented challenges when seeking to accurately test large numbers of individuals for SARS-CoV-2 antibody levels with a rapid turnaround time. ELISA platforms capable of serial dilution analysis coupled with a highly flexible software interface may provide a useful tool when seeking to define endpoint titers in a high-throughput manner. Immunohematology 2021;37:3343.
Assuntos
Tratamento Farmacológico da COVID-19 , COVID-19 , Anticorpos Antivirais , COVID-19/terapia , Ensaio de Imunoadsorção Enzimática , Humanos , Imunização Passiva , SARS-CoV-2 , Soroterapia para COVID-19RESUMO
BACKGROUND & OBJECTIVES: Red blood cells (RBCs) may be stored up to 42 days before transfusion, per US and EU standards. Although there is ample evidence that RBCs undergo deleterious changes during storage, studies assessing outcomes relative to storage time report conflicting findings. This study investigated RBC storage duration perspectives and practices among blood banking and transfusion professionals. MATERIALS & METHODS: A survey was administered at the American Association of Blood Banking annual meeting in October 2014 (N = 69). RESULTS: On average, participants believed RBC storage should not exceed 34 days (median: 35; range: 1-52), and estimated that RBCs are typically stored 21 days before transfusion at their institutions (median: 20; range: 10-40). There was 97% agreement that minimizing/reversing changes during RBC storage may produce clinical benefits; however, 80% believed the research does not consistently demonstrate worse outcomes using older blood. Two-thirds agreed that RBC storage duration is a major concern, but 81% agreed most institutions are not pursuing measures to shorten storage. CONCLUSIONS: This study found that many transfusion professionals believe RBCs should be stored for fewer than the 42 days currently allowed and that further efforts are warranted to abrogate changes in stored RBCs. These findings suggest a need for increased awareness of potential consequences of extended RBC storage and for strategies to maximize transfusion benefits.
Assuntos
Segurança do Sangue/normas , Transfusão de Eritrócitos/normas , Estudos Transversais , Eritrócitos/fisiologia , Pesquisas sobre Atenção à Saúde , Humanos , Fatores de Tempo , Resultado do TratamentoRESUMO
OBJECTIVE: To determine if billing records accurately report the receipt of red blood cell transfusions. BACKGROUND: Many red blood transfusions are given inappropriately, but efforts to monitor transfusion rates are hampered by a lack of data that facilitate benchmarking. METHODS: Using billing records and electronic medical records from 53 community hospitals, we estimated the sensitivity and specificity of billing records for measuring the receipt of transfusions in patients undergoing surgery for hip fractures. RESULTS: The sample included 12 091 patients, of whom 5215 received red blood cell transfusions according to electronic medical records. The sensitivity of billing data for measuring transfusions was 71·6% (95% CI: 60·4-82·8%). The specificity was 92·6% (95% CI: 88·3-97·0%). CONCLUSIONS: Researchers can use billing records to measure transfusion rates but should consider excluding hospitals with very low transfusion rates, which may indicate that the hospitals do not accurately report transfusions in billing data.
Assuntos
Transfusão de Eritrócitos , Revisão da Utilização de Seguros , Sistemas Computadorizados de Registros Médicos , Feminino , Humanos , MasculinoRESUMO
BACKGROUND AND OBJECTIVES: Human neutrophil antibodies (HNA) have been associated with severe transfusion-related acute lung injury (TRALI). We identified HNA antibodies in a blood donor population and performed an observational lookback on patients who received products from these donors to determine whether TRALI was associated with these transfusions. MATERIALS AND METHODS: Human neutrophil antibodies were determined in 1171 blood donors (388 non-transfused males, 390 human leucocyte antigen (HLA) antibody-negative females and 393 HLA antibody-positive females) for IgG and IgM antibodies using a flow cytometric assay. Selected positive samples had a monoclonal antibody immobilization of granulocyte antigen (MAIGA) and neutrophil genotyping performed to confirm specificity. Lookback was performed on patients receiving blood from donors with positive samples by extracting recipient data from hospital medical records. An expert panel of three pulmonary critical care physicians reviewed the summarized data and assigned a diagnosis of TRALI, possible TRALI, cannot distinguish between TRALI and TACO, TACO and other. RESULTS: Eight donors had HNA antibodies of which five contributed to this lookback (3-HNA-specific antibodies, 2-HNA non-specific antibodies). Seventy-six blood products were transfused from these donors into individual patients. One patient developed TRALI that was associated with a donor with a non-specific HNA antibody as well as class-I and class-II HLA antibodies. CONCLUSION: The incidence of TRALI in this lookback was low and combined with low frequency of HNA antibodies in the donor population suggests not screening donors for HNA antibodies at this time is acceptable.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/sangue , Doadores de Sangue , Antígenos HLA/sangue , Neutrófilos/imunologia , Anticorpos Anticitoplasma de Neutrófilos/imunologia , Feminino , Antígenos HLA/imunologia , Humanos , Masculino , Neutrófilos/químicaRESUMO
OBJECTIVES: Examine possible pooling strategies designed to expand SARS-CoV-2 serological testing capacity. METHODS: Negative pools were assessed to determine optimal optical density (OD) cutoffs, followed by spiking weak or strong positive samples to assess initial assay performance. Samples were then randomly subjected to pool and individual testing approaches. RESULTS: Single positive specimens consistently converted pools of 5, 10, or 20 into positive outcomes. However, weaker IgG-positive samples failed to similarly convert pools of 50 to a positive result. In contrast, a stronger individual positive sample converted all pools tested into positive outcomes. Finally, examination of 150 samples configured into pools of 5, 10, 20 or 50 accurately predicted the presence of positive or negative specimens within each pool. CONCLUSIONS: These results suggest that pooling strategies may allow expansion of serological testing capacity. While limitations exist, such strategies may aid in large-scale epidemiological screening or identification of optimal convalescent plasma donors.
Assuntos
Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G/sangue , SARS-CoV-2/imunologia , Manejo de Espécimes/métodos , COVID-19/sangue , Teste Sorológico para COVID-19/instrumentação , Ensaio de Imunoadsorção Enzimática/instrumentação , Humanos , Nefelometria e Turbidimetria , Fatores de TempoRESUMO
In pheochromocytoma (PC12) cells nerve growth factor (NGF) and epidermal growth factor (EGF) activate similar receptor tyrosine kinase signaling pathways but evoke strikingly different biological outcomes: NGF induces differentiation and EGF acts as a mitogen. A novel approach was developed for identifying transcription factor activities associated with NGF-activated, but not EGF-activated, signaling, using random oligonucleotide clones from a DNA recognition library to isolate specific DNA binding proteins from PC12 nuclear extracts. A protein complex from NGF-treated, but not EGF-treated, cells was identified that exhibits increased mobility and DNA binding activity in gel mobility shift assays. The binding complex was identified in supershift assays as Fra-2/JunD. The clones used as probes contain either AP-1 or cAMP response element binding (CREB) recognition elements. Time course experiments revealed further differences in NGF and EGF signaling in PC12 cells. NGF elicits a more delayed and sustained ERK phosphorylation than EGF, consistent with previous reports. Both growth factors transiently induce c-fos, but NGF evokes a greater response than EGF. NGF specifically increases Fra-1 and Fra-2 levels at 4 and 24 hr. The latter is represented in Western blots by bands in the 40-46 kDa range. NGF, but not EGF, enhances the upper bands, corresponding to phosphorylated Fra-2. These findings suggest that prolonged alterations in Fra-2 and subsequent increases in Fra-2/JunD binding to AP-1 and CREB response elements common among many gene promoters could serve to trigger broadly an NGF-specific program of gene expression.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Neural/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/biossíntese , Animais , Sítios de Ligação/genética , Diferenciação Celular/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Antígeno 2 Relacionado a Fos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutagênese Sítio-Dirigida , Fator de Crescimento Neural/farmacologia , Células PC12/citologia , Células PC12/efeitos dos fármacos , Células PC12/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Sequências Reguladoras de Ácido Nucleico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Transcrição AP-1/metabolismoRESUMO
Septal neurons from embryonic rats were grown in tissue culture. Microfluorimetric and electrophysiological techniques were used to study Ca2+ homeostasis in these neurons. The estimated basal intracellular free ionized calcium concentration ([Ca2+]i) in the neurons was low (50-100 nM). Depolarization of the neurons with 50 mM K+ resulted in rapid elevation of [Ca2+]i to 500-1,000 nM showing recovery to baseline [Ca2+]i over several minutes. The increases in [Ca2+]i caused by K+ depolarization were completely abolished by the removal of extracellular Ca2+, and were reduced by approximately 80% by the 'L-type' Ca2+ channel blocker, nimodipine (1 microM). [Ca2+]i was also increased by the excitatory amino acid L-glutamate, quisqualate, AMPA and kainate. Responses to AMPA and kainate were blocked by CNQX and DNQX. In the absence of extracellular Mg2+, large fluctuations in [Ca2+]i were observed that were blocked by removal of extracellular Ca2+, by tetrodotoxin (TTX), or by antagonists of N-methyl D-aspartate (NMDA) such as 2-amino 5-phosphonovalerate (APV). In zero Mg2+ and TTX, NMDA caused dose-dependent increases in [Ca2+]i that were blocked by APV. Caffeine (10 mM) caused transient increases in [Ca2+]i in the absence of extracellular Ca2+, which were prevented by thapsigargin, suggesting the existence of caffeine-sensitive ATP-dependent intracellular Ca2+ stores. Thapsigargin (2 microM) had little effect on [Ca2+]i, or on the recovery from K+ depolarization. Removal of extracellular Na+ had little effect on basal [Ca2+]i or on responses to high K+, suggesting that Na+/Ca2+ exchange mechanisms do not play a significant role in the short-term control of [Ca2+]i in septal neurons. The mitochondrial uncoupler, CCCP, caused a slowly developing increase in basal [Ca2+]i; however, [Ca2+]i recovered as normal from high K+ stimulation in the presence of CCCP, which suggests that the mitochondria are not involved in the rapid buffering of moderate increases in [Ca2+]i. In simultaneous electrophysiological and microfluorimetric recordings, the increase in [Ca2+]i associated with action potential activity was measured. The amplitude of the [Ca2+]i increase induced by a train of action potentials increased with the duration of the train, and with the frequency of firing, over a range of frequencies between 5 and 200 Hz. Recovery of [Ca2+]i from the modest Ca2+ loads imposed on the neuron by action potential trains follows a simple exponential decay (tau = 3-5 s).
Assuntos
Encéfalo/fisiologia , Cálcio/metabolismo , Homeostase/fisiologia , Neurônios/metabolismo , Aminoácidos/farmacologia , Animais , Encéfalo/citologia , Soluções Tampão , Técnicas de Cultura , Estimulação Elétrica , Eletrofisiologia , Feminino , Fluorometria , Fura-2 , Fármacos Neuromusculares Despolarizantes/farmacologia , Cloreto de Potássio/farmacologia , Gravidez , Ratos , Receptores de Glutamato/efeitos dos fármacosRESUMO
A culture system enriched for nerve growth factor (NGF) receptor bearing cells was developed to investigate signal transduction events activated by NGF in postmitotic central nervous system neurons. Cells from the septal region of embryonic rats at 16 days of gestation were grown on glass coverslips above a glial cell layer established from postnatal rat cortex. The separation of glial and neuronal planes in this "bilaminar" system permits the diffusion of glial-derived factors required by septal neurons for survival yet allows the investigation of NGF responses in a pure neuronal population. Approximately 15% of the neurons in this culture system were immunoreactive for the low affinity NGF receptor. NGF rapidly increased MAP kinase activity (2-5 min) and transiently induced expression of c-fos in septal neurons. NGF treatment also increased choline acetyltransferase activity, while the number of cholinergic neurons remained constant. Septal neuron survival depended on the presence of glial cells, but neuronal viability in the bilaminar system was unaffected by anti-NGF antiserum, indicating that glial-derived neurotrophic support is not mediated by NGF alone. These data suggest that the bilaminar culture system is a useful system for the study of early events in NGF-activated signal transduction and the nature of glial-derived trophic support of developing basal forebrain neurons.
Assuntos
Expressão Gênica/efeitos dos fármacos , Fatores de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Septo Pelúcido/embriologia , Transdução de Sinais/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Idade Gestacional , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/efeitos dos fármacos , Neurônios/citologia , Neurotransmissores/metabolismo , Fenótipo , Fosforilação , Ratos , Septo Pelúcido/efeitos dos fármacosRESUMO
Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are members of a family of trophic factors designated the neurotrophins, each of which can bind to the low-affinity NGF receptor (LNGFR). To investigate the mechanisms that regulate the expression of the neurotrophins and the LNGFR in the developing brain, we grew cells from the embryonic mouse septum and hippocampus in reaggregating cell culture and compared neurotrophin and LNGFR expression in developing reaggregates with that seen in the developing septum and hippocampus in situ. NGF, BDNF, NT-3 and LNGFR were each expressed in septal and hippocampal reaggregates as well as the native septum and hippocampus. Additionally, the temporal expression profiles observed in reaggregates were generally similar to those seen in the respective brain regions in situ. In order to determine whether NGF can modulate neurotrophin or LNGFR expression, reaggregates were cultured in the continual presence of either exogenous NGF or anti-NGF antibodies. NGF-treated septal cultures expressed twice the level of LNGFR mRNA as was seen in untreated septal cultures; on the other hand, septal cultures grown in the presence of anti-NGF antibodies, to neutralize endogenously synthesized NGF, displayed a 3-fold decrease in LNGFR mRNA expression compared to untreated cultures. No effects of NGF or anti-NGF were observed on LNGFR expression in hippocampal reaggregates, or on neurotrophin mRNA expression in either reaggregate type. These results suggest that regulatory mechanisms intrinsic to the septal and hippocampal regions control neurotrophin and LNGFR expression. NGF is likely to be one of these regulatory cues since it acts locally in septal reaggregates to control the developmental expression of LNGFR mRNA. The possible roles of locally synthesized NGF and other neurotrophins in the development of septal neurons are discussed.
Assuntos
Envelhecimento/fisiologia , Encéfalo/fisiologia , Hipocampo/fisiologia , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Receptores de Fator de Crescimento Neural/metabolismo , Animais , Encéfalo/citologia , Fator Neurotrófico Derivado do Encéfalo , Agregação Celular , Células Cultivadas , Córtex Cerebral/fisiologia , Embrião de Mamíferos , Ensaio de Imunoadsorção Enzimática , Idade Gestacional , Hipocampo/citologia , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/fisiologia , Neurotrofina 3 , RNA Mensageiro/metabolismoRESUMO
The basal forebrain has attracted considerable attention because of its putative role in complex functions such as learning, memory and behavioral state control as well as its vulnerability in neurological disorders such as Alzheimer's Disease (AD). The finding that nerve growth factor provides trophic support for the cholinergic basal forebrain neurons has stimulated further interest in understanding trophic interactions of basal forebrain neurons as well as in possible trophic factor therapeutic strategies for disease states. Our laboratory has utilized primary cell cultures and developed immortalized central nervous system cell lines to study the trophic interactions that establish and maintain the septohippocampal pathway, a basal forebrain component which plays an essential role in cognitive function and is prominently affected in AD. The results of our primary cell culture studies have demonstrated the importance of trophic signals elaborated by the hippocampus in mediating the development of septal cholinergic neurons. Nerve growth factor plays an important role in this process, but it cannot account for all of the trophic signals elaborated by authentic hippocampal target cells. The development by this laboratory of clonal cell lines of septal and hippocampal lineage offers the prospect of investigating both the response to and elaboration of neural trophic signals at a more precise level of resolution than can be achieved with primary cultures. The technology and information that is generated from the engineering of such cell lines will also serve as a strategy to study trophic interactions in other brain circuits in future years, and to investigate possible changes or dysfunctions that occur neurological diseases.
Assuntos
Neurônios/fisiologia , Prosencéfalo/fisiologia , Doença de Alzheimer/fisiopatologia , Animais , Encéfalo/fisiologia , Encéfalo/fisiopatologia , Células Cultivadas , Células Clonais , Técnicas de Cultura/métodos , Humanos , Modelos Neurológicos , Fatores de Crescimento Neural/fisiologiaAssuntos
Acetilcolina/fisiologia , Vias Aferentes/anatomia & histologia , Comportamento/fisiologia , Fibras Colinérgicas/ultraestrutura , Cognição/fisiologia , Doenças do Sistema Nervoso/fisiopatologia , Vias Aferentes/fisiologia , Animais , Mapeamento Encefálico , Córtex Cerebral/anatomia & histologia , Córtex Cerebral/fisiologia , Fibras Colinérgicas/fisiologia , Hipocampo/anatomia & histologia , Hipocampo/fisiologia , Humanos , Modelos Biológicos , Neurotransmissores/fisiologia , Primatas , Prosencéfalo/anatomia & histologia , Prosencéfalo/fisiologia , Ratos , Sono/fisiologia , Tegmento Mesencefálico/anatomia & histologia , Tegmento Mesencefálico/fisiologiaAssuntos
Infecções Oportunistas Relacionadas com a AIDS/imunologia , Transfusão de Sangue , Soropositividade para HIV/terapia , HIV-1/imunologia , HIV-2/imunologia , Ativação Viral/imunologia , Infecções Oportunistas Relacionadas com a AIDS/virologia , Animais , Infecções por Citomegalovirus/imunologia , Infecções por Herpesviridae/imunologia , Herpesvirus Humano 6/imunologia , Herpesvirus Humano 7/imunologia , Herpesvirus Humano 8/imunologia , HumanosRESUMO
BACKGROUND: Specific subsets of peripheral blood WBCs are reservoirs for infectious agents, such as CMV and EBV, and can serve as vectors for transfusion transmission of these agents. While filter WBC reduction has been used to prevent transfusion transmission of infections, its effectiveness has not been documented for many infectious agents and in some instances may be difficult to demonstrate in clinical trials. Because the effectiveness of filtration depends on the number of infected WBCs remaining at transfusion, WBC subpopulations in packed RBC units were quantitated after filtration and storage. STUDY DESIGN AND METHODS: Packed RBC units (n = 14) were filtered and stored at 4(o)C for 42 days or were stored without filtration. Serial samples were subjected to flow cytometric immunophenotyping of WBC subsets: neutrophils, monocytes, CD4+ and CD8+ T cells, B cells, and NK cells. RESULTS: Filtration produced a mean reduction in total WBCs of 3.2 log. Monocytes, lymphocytes, and neutrophils were reduced by 4.1, 3.8, and 2.5 log, respectively. Lymphocyte subsets also demonstrated differential reduction with filtration. All WBC subsets showed ongoing loss during storage. CONCLUSIONS: Monocyte and lymphocyte subsets are removed most effectively by prestorage filtration. Postfiltration storage leads to further significant reductions in WBC subsets. The implications of these findings for the mitigation of transfusion transmission of infection are discussed.
Assuntos
Infecções por Citomegalovirus/transmissão , Filtração , Hematócrito , Infecções por Herpesviridae/transmissão , Herpesvirus Humano 4 , Herpesvirus Humano 8 , Leucócitos/classificação , Anticorpos , Antígenos CD13/sangue , Infecções por Citomegalovirus/imunologia , Citometria de Fluxo , Humanos , Antígenos Comuns de Leucócito/imunologia , Estudos Longitudinais , Linfócitos/citologia , Monócitos/citologia , Neutrófilos/citologia , Reação TransfusionalRESUMO
The present study examined the effects of removing hippocampal nerve growth factor (NGF)-producing neurons upon cholinergic and noncholinergic septohippocampal projecting neurons. To deplete septal/diagonal band neurons of their intrinsic source of NGF, rats received unilateral intrahippocampal injections of ibotenic acid and were sacrificed 2-24 weeks later. Choline acetyltransferase and parvalbumin immunohistochemistry failed to reveal changes in the number of cholinergic or gamma-aminobutyric acid-containing neurons, respectively, within the septal/diagonal band region ipsilateral to the hippocampal lesion at any time point examined. Additionally, immunocytochemical localization of nonphosphorylated and phosphorylated neurofilament proteins did not reveal abnormal staining characteristics within the septal/diagonal band complex, suggesting that this lesion does not alter cytoskeletal features of neurons which project to the hippocampus. Selected rats received unilateral hippocampal lesions and 3 months later were injected with fluorogold into the remaining hippocampal remnant and with wheat germ agglutinin conjugated to horse radish peroxidase into the intact contralateral hippocampus. Both retrograde tracers were predominantly transported to their respective ipsilateral septum and vertical limb of the diagonal band. This indicates that following the lesion, septal/diagonal band neurons still project ipsilaterally and sprouting to the NGF-rich contralateral side does not occur. RNA blot analysis revealed a decrease in NGF mRNA expression within the lesioned hippocampus with a maximum reduction of approximately 70%. In contrast, no change in NGF mRNA expression was observed within the ipsilateral septum relative to the contralateral side. The present study demonstrates that removal of hippocampal target neurons does not alter the number, morphology, or projections of both cholinergic and noncholinergic septal/diagonal band neurons.
Assuntos
Colina O-Acetiltransferase/metabolismo , Hipocampo/fisiologia , Ácido Ibotênico/toxicidade , Fatores de Crescimento Neural/biossíntese , Animais , Transporte Axonal , Northern Blotting , Feminino , Lateralidade Funcional , Expressão Gênica , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Peroxidase do Rábano Silvestre , Imuno-Histoquímica , Fatores de Crescimento Neural/genética , Proteínas de Neurofilamentos/análise , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Parvalbuminas/análise , Parvalbuminas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos , Conjugado Aglutinina do Germe de Trigo-Peroxidase do Rábano Silvestre , Aglutininas do Germe de TrigoRESUMO
Nerve growth factor (NGF) is synthesized in the hippocampus and neocortex and provides trophic support for afferent cholinergic neurons of the basal forebrain. To determine the capacity of the developing hippocampus to express NGF in the absence of NGF-responsive afferents, embryonic hippocampal cells isolated prior to septal innervation were studied in reaggregating cell culture. The expression of NGF protein in vitro was qualitatively and quantitatively similar to that observed in situ. The expression of NGF mRNA exhibited an initial increase in vitro but then plateaued and was maintained at a steady level. This latter finding was in contrast to the steady rise in NGF mRNA levels observed in situ. These data suggest that (i) intrinsic hippocampal interactions regulate the onset of NGF expression, but that (ii) additional extrinsic developmental signals may be required for proper regulation of hippocampal NGF expression during ontogeny.
Assuntos
Hipocampo/crescimento & desenvolvimento , Fatores de Crescimento Neural/genética , Envelhecimento , Animais , Agregação Celular , Células Cultivadas , Expressão Gênica , Regulação da Expressão Gênica , Hipocampo/citologia , Hipocampo/metabolismo , Fatores de Crescimento Neural/biossíntese , RNA/isolamento & purificação , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , RatosRESUMO
MN9D is an immortalized dopamine-containing neuronal hybrid cell line. When MN9D cells were coaggregated with primary embryonic cells of optic tectum, a brain region that does not receive a dopaminergic innervation, there was a marked reduction in their dopamine content, tyrosine hydroxylase immunoreactivity, and tyrosine hydroxylase mRNA. Similar reductions in dopamine content were produced by coaggregation with cells from embryonic thalamus, another brain region devoid of dopaminergic innervation. Coaggregation of MN9D cells with dopaminoceptive cells from the corpus striatum or the cortex did not have a demonstrable stimulatory effect on the dopamine content of MN9D cells. The decrease in MN9D dopamine content produced by optic tectum cells was not reversed by addition of corpus striatum cells. Thus, the MN9D hybrid cells are able to respond to an inhibitory factor(s) from cells derived from brain areas that are not targets for dopaminergic neurons. Catecholamine-producing PC12 cells did not respond in a similar manner, suggesting that the response of MN9D cells is a function of their mesencephalic origin. Given the selective response of MN9D cells to different brain cell populations, this hybrid cell line should facilitate investigations of cell-cell interactions in the central nervous system that may be involved in the expression of neurotransmitter phenotype and establishment of specific neuronal connections.
Assuntos
Encéfalo/metabolismo , Dopamina/metabolismo , Neurônios/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Animais , Encéfalo/citologia , Agregação Celular , Linhagem Celular , Colina O-Acetiltransferase/metabolismo , Dopamina/biossíntese , Embrião de Mamíferos , Células Híbridas/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Neurônios/citologia , Especificidade de Órgãos , Tirosina 3-Mono-Oxigenase/análiseRESUMO
Cortical glial cells in culture were found to be responsive to the neurotrophin brain-derived neurotrophic factor (BDNF), as evidenced by activation of multiple signal transduction processes. BDNF produced an increase in mitogen-activated protein (MAP) kinase tyrosine phosphorylation, MAP kinase activity, intracellular calcium concentration and c-fos expression in the glial cells. Only a subset of the glial cells responded to BDNF, as reflected in single-cell analysis of calcium transients and c-fos expression. BDNF had no detectable effect on glial mitotic activity, as measured by DNA synthesis. In parallel studies, nerve growth factor and neurotrophin-3 had no effect on signalling in these cultures. BDNF has previously been demonstrated to act via trkB receptors with a cytoplasmic tyrosine kinase domain (gp145trkB). Pretreatment of glial cultures with K252a, which at low concentrations specifically inhibits the trk tyrosine kinases, abolished BDNF effects on MAP kinase stimulation, suggesting that BDNF was acting through gp145trkB. However, subsequent studies showed that gp145trkB was expressed at extremely low levels in the cultures: gp145trkB mRNA transcripts could only be detected using the reverse transcription-polymerase chain reaction, and gp145trkB protein was not detected by either immunoblotting or immunocytochemistry. On the other hand, the glia expressed significantly higher levels of gp95trkB mRNA and protein, which represent truncated forms of trkB receptors lacking the tyrosine kinase domain. The results of these studies demonstrate that a subset of cultured CNS glia respond to BDNF with the activation of conventional signal transduction processes. The mechanism of BDNF-initiated signal transduction in glial cells most likely involves a relatively small number of gp145trkB receptors, but involvement of the more abundant truncated gp95trkB receptors cannot be excluded.
Assuntos
Proteínas do Tecido Nervoso/farmacologia , Neuroglia/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Fator Neurotrófico Derivado do Encéfalo , Cálcio/metabolismo , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Expressão Gênica , Imuno-Histoquímica , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
Clonal cell lines of rat embryonic hippocampal origin have been developed by using retroviral transduction of temperature-sensitive simian virus 40 large tumor antigens. The cell lines undergo morphological differentiation at the nonpermissive temperature and in response to differentiating agents. Immunocytochemical analysis indicates that various lines are derived from progenitors of neuronal, glial, and bipotential lineages. Selected neuronal lines differentiate in response to diffusible factors released by primary glia, and one line of glial lineage supports the maturation of primary neurons in culture. Selected cell lines exhibit different patterns of neurotrophin gene expression that change after differentiation. In some lines, the relative levels of neurotrophin 3 and brain-derived neurotrophic factor message expression may reflect the developmental or regional differential expression seen for these genes in the hippocampus in situ. These hippocampal cell lines, which express markers indicative of commitment to neuronal or glial lineages, are valuable for studies of development and plasticity in these lineages, as well as for studies of the regulation of neural trophic interactions.
Assuntos
Transformação Celular Viral , Hipocampo/fisiologia , Proteínas do Tecido Nervoso/genética , Neuroglia/fisiologia , Neurônios/fisiologia , Vírus 40 dos Símios/genética , Células 3T3 , Animais , Antígenos Transformantes de Poliomavirus/genética , Fator Neurotrófico Derivado do Encéfalo , Linhagem Celular Transformada , Células Cultivadas , Células Clonais , Replicação do DNA , Embrião de Mamíferos , Expressão Gênica , Camundongos , Fatores de Crescimento Neural/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Timidina/metabolismoRESUMO
CD40 ligand (CD40L), expressed on activated T cells, binds its receptor, CD40, on dendritic cells, B cells, and monocytes/ macrophages. Human immunodeficiency virus (HIV)-infected individuals exhibit normal B-cell CD40 expression but diminished expression of CD40L on CD4 + T cells. Thus, we studied recombinant human CD40L (huCD40L) in an in vitro rhesus macaque model of acquired immunodeficiency syndrome (AIDS). huCD40L induced peripheral blood mononuclear cell (PBMC) proliferation independent of mitogenic cytokines and led to a 70% reduction in p27 production by simian immunodeficiency virus (SIV) mac239 infected PBMCs (P < 0.05). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed reduced expression of SIV gag and increased expression of interleukin (IL)-16 mRNA. Supernatants from huCD40L-stimulated PBMC and control cultures contained similar amounts of IL-16, suggesting an intracellular antiviral effect by IL-16. Phytohemagglutinin (PHA)-stimulated PBMCs similarly cultured with huCD40L showed only slight increases in chemokine production (P > 0.05). These results suggest that huCD40L inhibits replication (antigen and mRNA production) of SIVmac239. This response involves huCD40L induction of IL16 mRNA expression and appears to be independent of beta-chemokines.
Assuntos
Regulação Viral da Expressão Gênica , Interleucina-16/farmacologia , Glicoproteínas de Membrana/farmacologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/fisiologia , Animais , Ligante de CD40 , Citocinas/imunologia , Modelos Animais de Doenças , Humanos , Interleucina-16/biossíntese , Interleucina-16/imunologia , Macaca mulatta/imunologia , Glicoproteínas de Membrana/imunologia , Monócitos/imunologia , RNA Mensageiro/análise , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Síndrome de Imunodeficiência Adquirida dos Símios/fisiopatologia , Vírus da Imunodeficiência Símia/genética , Replicação Viral/efeitos dos fármacosRESUMO
The hippocampal formation elaborates trophic factors such as nerve growth factor (NGF) to support the cholinergic innervation it receives from the septal region. To further study the trophic interactions of this pathway, hippocampal cells from embryonic day 18 and postnatal day 21 mice were immortalized via somatic cell fusion to N18TG2 neuroblastoma cells. The hippocampal cell lines exhibit morphological and cytoskeletal features which are typical of their neuronal parents but which are not expressed by the neuroblastoma parent. When differentiated with retinoic acid, the hippocampal cell lines exhibit electrophysiological features similar to cultured hippocampal neurons. Many of the lines constitutively express high levels of NGF, and at least one cell line exerts a non-NGF trophic effect on the expression of choline acetyltransferase by septal neurons in vitro. These cell lines are potentially useful for investigating the neurochemical and excitable properties of hippocampal neurons and identifying novel trophic activities that promote the development and maintenance of the septohippocampal pathway.