RESUMO
In vitro studies have shown that deletion of nef and deleterious mutation in the Nef dimerization interface attenuates HIV replication and associated pathogenesis. Humanized rodents with human immune cells and lymphoid tissues are robust in vivo models for investigating the interactions between HIV and the human immune system. Here, we demonstrate that nef deletion impairs HIV replication and HIV-induced immune dysregulation in the blood and human secondary lymphoid tissue (human spleen) in bone marrow-liver-thymus-spleen (BLTS) humanized mice. Furthermore, we also show that nef defects (via deleterious mutations in the dimerization interface) impair HIV replication and HIV-induced immune dysregulation in the blood and human spleen in BLTS-humanized mice. We demonstrate that the reduced replication of nef-deleted and nef-defective HIV is associated with robust antiviral innate immune response, and T helper 1 response. Our results support the proposition that Nef may be a therapeutic target for adjuvants in HIV cure strategies.
Assuntos
Modelos Animais de Doenças , Infecções por HIV , HIV-1 , Fígado , Baço , Viremia , Replicação Viral , Produtos do Gene nef do Vírus da Imunodeficiência Humana , Animais , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Camundongos , Humanos , Viremia/imunologia , Baço/imunologia , Baço/virologia , HIV-1/imunologia , HIV-1/genética , HIV-1/fisiologia , Fígado/virologia , Fígado/imunologia , Fígado/patologia , Medula Óssea/virologia , Medula Óssea/imunologia , Timo/imunologia , Timo/virologia , Imunidade InataRESUMO
Generation of a stable long-lived plasma cell (LLPC) population is the sine qua non of durable antibody responses after vaccination or infection. We studied 20 individuals with a prior coronavirus disease 2019 infection and characterized the antibody response using bone marrow aspiration and plasma samples. We noted deficient generation of spike-specific LLPCs in the bone marrow after severe acute respiratory syndrome coronavirus 2 infection. Furthermore, while the regression model explained 98% of the observed variance in anti-tetanus immunoglobulin G levels based on LLPC enzyme-linked immunospot assay, we were unable to fit the same model with anti-spike antibodies, again pointing to the lack of LLPC contribution to circulating anti-spike antibodies.
Assuntos
Anticorpos Antivirais , Medula Óssea , COVID-19 , Plasmócitos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Humanos , COVID-19/imunologia , Plasmócitos/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Antivirais/sangue , SARS-CoV-2/imunologia , Masculino , Pessoa de Meia-Idade , Feminino , Medula Óssea/virologia , Adulto , Imunoglobulina G/sangue , IdosoRESUMO
Hematopoiesis is a tightly regulated process that gets skewed toward myelopoiesis. This restrains lymphopoiesis, but the role of lymphocytes in this process is not well defined. To unravel the intricacies of neutrophil responses in COVID-19, we performed bulk RNAseq on neutrophils from healthy controls and COVID-19 patients. Principal component analysis revealed distinguishing neutrophil gene expression alterations in COVID-19 patients. ICU and ward patients displayed substantial transcriptional changes, with ICU patients exhibiting a more pronounced response. Intriguingly, neutrophils from COVID-19 patients, notably ICU patients, exhibited an enrichment of immunoglobulin (Ig) and B cell lineage-associated genes, suggesting potential lineage plasticity. We validated our RNAseq findings in a larger cohort. Moreover, by reanalyzing single-cell RNA sequencing (scRNAseq) data on human bone marrow (BM) granulocytes, we identified the cluster of granulocyte-monocyte progenitors (GMP) enriched with Ig and B cell lineage-associated genes. These cells with lineage plasticity may serve as a resource depending on the host's needs during severe systemic infection. This distinct B cell subset may play a pivotal role in promoting myelopoiesis in response to infection. The scRNAseq analysis of BM neutrophils in infected mice further supported our observations in humans. Finally, our studies using an animal model of acute infection implicate IL-7/GM-CSF in influencing neutrophil and B cell dynamics. Elevated GM-CSF and reduced IL-7 receptor expression in COVID-19 patients imply altered hematopoiesis favoring myeloid cells over B cells. Our findings provide novel insights into the relationship between the B-neutrophil lineages during severe infection, hinting at potential implications for disease pathogenesis. IMPORTANCE: This study investigates the dynamics of hematopoiesis in COVID-19, focusing on neutrophil responses. Through RNA sequencing of neutrophils from healthy controls and COVID-19 patients, distinct gene expression alterations are identified, particularly in ICU patients. Notably, neutrophils from COVID-19 patients, especially in the ICU, exhibit enrichment of immunoglobulin and B cell lineage-associated genes, suggesting potential lineage plasticity. Validation in a larger patient cohort and single-cell analysis of bone marrow granulocytes support the presence of granulocyte-monocyte progenitors with B cell lineage-associated genes. The findings propose a link between B-neutrophil lineages during severe infection, implicating a potential role for these cells in altered hematopoiesis favoring myeloid cells over B cells. Elevated GM-CSF and reduced IL-7 receptor expression in stress hematopoiesis suggest cytokine involvement in these dynamics, providing novel insights into disease pathogenesis.
Assuntos
COVID-19 , Hematopoese , Neutrófilos , Humanos , Animais , COVID-19/imunologia , COVID-19/virologia , Camundongos , Neutrófilos/imunologia , Neutrófilos/virologia , Linfócitos B/imunologia , Linfócitos B/virologia , SARS-CoV-2/genética , Medula Óssea/virologia , Linhagem da Célula , Masculino , Feminino , Pessoa de Meia-Idade , Análise de Célula Única , Mielopoese/genética , Análise de Sequência de RNARESUMO
HIV is difficult to eradicate due to the persistence of a long-lived reservoir of latently infected cells. Previous studies have shown that natural killer cells are important to inhibiting HIV infection, but it is unclear whether the administration of natural killer cells can reduce rebound viremia when anti-retroviral therapy is discontinued. Here we show the administration of allogeneic human peripheral blood natural killer cells delays viral rebound following interruption of anti-retroviral therapy in humanized mice infected with HIV-1. Utilizing genetically barcoded virus technology, we show these natural killer cells efficiently reduced viral clones rebounding from latency. Moreover, a kick and kill strategy comprised of the protein kinase C modulator and latency reversing agent SUW133 and allogeneic human peripheral blood natural killer cells during anti-retroviral therapy eliminated the viral reservoir in a subset of mice. Therefore, combinations utilizing latency reversal agents with targeted cellular killing agents may be an effective approach to eradicating the viral reservoir.
Assuntos
Fármacos Anti-HIV/farmacologia , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/terapia , HIV-1/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Inibidores de Proteínas Quinases/farmacologia , Viremia/terapia , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/imunologia , Medula Óssea/virologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Técnicas de Cocultura , Feminino , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/imunologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Células Matadoras Naturais/transplante , Masculino , Camundongos , Camundongos Transgênicos , Proteína Quinase C/genética , Proteína Quinase C/imunologia , Baço/efeitos dos fármacos , Baço/imunologia , Baço/virologia , Carga Viral/efeitos dos fármacos , Viremia/genética , Viremia/imunologia , Viremia/virologia , Latência Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Determining how hematopoietic stem and progenitor cells (HSPCs) can be infected by viruses is necessary to understand and predict how the immune system will drive the host response. We present here a protocol to analyze the capacity of SARS-CoV-2 to infect different subsets of human HSPCs, inlcuding procedures for SARS-CoV-2 production and titration, isolation of human HSPCs from different sources (bone marrow, umbilical cord, or peripheral blood), and quantification of SARS-Cov-2 infection capacity by RT-qPCR and colony forming unit assay. For complete details on the use and execution of this protocol, please refer to Huerga Encabo et al. (2021).
Assuntos
Medula Óssea/virologia , Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/virologia , Ensaio de Unidades Formadoras de Colônias/métodos , Sangue Fetal/virologia , Células-Tronco Hematopoéticas/virologia , SARS-CoV-2/isolamento & purificação , COVID-19/patologia , Células-Tronco Hematopoéticas/patologia , HumanosRESUMO
Chronic viral infections are associated with hematopoietic suppression, bone marrow (BM) failure, and hematopoietic stem cell (HSC) exhaustion. However, how persistent viral challenge and inflammatory responses target BM tissues and perturb hematopoietic competence remains poorly understood. Here, we combine functional analyses with advanced 3D microscopy to demonstrate that chronic infection with lymphocytic choriomeningitis virus leads to (1) long-lasting decimation of the BM stromal network of mesenchymal CXCL12-abundant reticular cells, (2) proinflammatory transcriptional remodeling of remaining components of this key niche subset, and (3) durable functional defects and decreased competitive fitness in HSCs. Mechanistically, BM immunopathology is elicited by virus-specific, activated CD8 T cells, which accumulate in the BM via interferon-dependent mechanisms. Combined antibody-mediated inhibition of type I and II IFN pathways completely preempts degeneration of CARc and protects HSCs from chronic dysfunction. Hence, viral infections and ensuing immune reactions durably impact BM homeostasis by persistently decreasing the competitive fitness of HSCs and disrupting essential stromal-derived, hematopoietic-supporting cues.
Assuntos
Medula Óssea/virologia , Células-Tronco Hematopoéticas/patologia , Células-Tronco Hematopoéticas/virologia , Coriomeningite Linfocítica/patologia , Animais , Medula Óssea/metabolismo , Medula Óssea/patologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/virologia , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Doença Crônica , Regulação da Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Interferons/metabolismo , Coriomeningite Linfocítica/metabolismo , Coriomeningite Linfocítica/virologia , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/imunologia , Receptor de Interferon alfa e beta/metabolismoRESUMO
Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tickborne infectious disease in China, Korea, and Japan caused by the SFTS virus (SFTSV). SFTS has a high mortality rate due to multiorgan failure. Recently, there are several reports on SFTS patients with mycosis. Here, we report a middle-aged Japanese SFTS patient with invasive pulmonary aspergillosis (IPA) revealed by an autopsy. A 61-year-old man with hypertension working in forestry was bitten by a tick and developed fever, diarrhea, and anorexia in 2 days. On day 4, consciousness disorder was appearing, and the patient was transferred to the University of Miyazaki Hospital. A blood test showed leukocytopenia, thrombocytopenia, as well as elevated levels of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and creatine kinase. The SFTSV gene was detected in serum using a reverse-transcription polymerase chain reaction. On day 5, respiratory failure appeared and progressed rapidly, and on day 7, the patient died. An autopsy was performed that revealed hemophagocytosis in the bone marrow and bleeding of several organs. IPA was observed in lung specimens. SFTSV infection may be a risk factor for developing IPA. Early diagnosis and treatment of IPA may be important in patients with SFTS.
Assuntos
Aspergilose Pulmonar Invasiva/diagnóstico por imagem , Aspergilose Pulmonar Invasiva/virologia , Phlebovirus/patogenicidade , Febre Grave com Síndrome de Trombocitopenia/complicações , Animais , Autopsia , Medula Óssea/virologia , Evolução Fatal , Humanos , Aspergilose Pulmonar Invasiva/microbiologia , Japão , Pulmão/patologia , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Febre Grave com Síndrome de Trombocitopenia/diagnóstico , Trombocitopenia/etiologia , Doenças Transmitidas por Carrapatos/transmissão , Doenças Transmitidas por Carrapatos/virologia , Carrapatos/virologiaRESUMO
BACKGROUND: Multiple myeloma (MM) remains an incurable disease and oncolytic viruses offer a well-tolerated addition to the therapeutic arsenal. Oncolytic reovirus has progressed to phase I clinical trials and its direct lytic potential has been extensively studied. However, to date, the role for reovirus-induced immunotherapy against MM, and the impact of the bone marrow (BM) niche, have not been reported. METHODS: This study used human peripheral blood mononuclear cells from healthy donors and in vitro co-culture of MM cells and BM stromal cells to recapitulate the resistant BM niche. Additionally, the 5TGM1-Kalw/RijHSD immunocompetent in vivo model was used to examine reovirus efficacy and characterize reovirus-induced immune responses in the BM and spleen following intravenous administration. Collectively, these in vitro and in vivo models were used to characterize the development of innate and adaptive antimyeloma immunity following reovirus treatment. RESULTS: Using the 5TGM1-Kalw/RijHSD immunocompetent in vivo model we have demonstrated that reovirus reduces both MM tumor burden and myeloma-induced bone disease. Furthermore, detailed immune characterization revealed that reovirus: (i) increased natural killer (NK) cell and CD8+ T cell numbers; (ii) activated NK cells and CD8+ T cells and (iii) upregulated effector-memory CD8+ T cells. Moreover, increased effector-memory CD8+ T cells correlated with decreased tumor burden. Next, we explored the potential for reovirus-induced immunotherapy using human co-culture models to mimic the myeloma-supportive BM niche. MM cells co-cultured with BM stromal cells displayed resistance to reovirus-induced oncolysis and bystander cytokine-killing but remained susceptible to killing by reovirus-activated NK cells and MM-specific cytotoxic T lymphocytes. CONCLUSION: These data highlight the importance of reovirus-induced immunotherapy for targeting MM cells within the BM niche and suggest that combination with agents which boost antitumor immune responses should be a priority.
Assuntos
Medula Óssea/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Matadoras Naturais/imunologia , Mieloma Múltiplo/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos/imunologia , Reoviridae/imunologia , Baço/imunologia , Microambiente Tumoral/imunologia , Animais , Medula Óssea/virologia , Linfócitos T CD8-Positivos/virologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Citocinas/imunologia , Citotoxicidade Imunológica , Feminino , Humanos , Células Matadoras Naturais/virologia , Masculino , Camundongos Endogâmicos C57BL , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/virologia , Vírus Oncolíticos/patogenicidade , Reoviridae/patogenicidade , Baço/virologia , Evasão TumoralAssuntos
Medula Óssea/virologia , COVID-19/complicações , Pancitopenia/etiologia , SARS-CoV-2/patogenicidade , Macroglobulinemia de Waldenstrom/complicações , Medula Óssea/patologia , COVID-19/patologia , COVID-19/virologia , Evolução Fatal , Feminino , Humanos , Pessoa de Meia-Idade , Pancitopenia/patologia , Pancitopenia/virologia , SARS-CoV-2/isolamento & purificação , Macroglobulinemia de Waldenstrom/patologia , Macroglobulinemia de Waldenstrom/virologiaRESUMO
BACKGROUND: Osteosarcoma is a malignant mesenchymal bone tumor. Although it is a common tumor in the appendicular skeleton of dogs and cats, it is rarely reported in birds. Retroviruses are usually associated with solid tumor development in different avian species. CASE PRESENTATION: This report aims to describe a case of osteosarcoma associated with the avian leukosis virus in a captive bare-faced curassow (Crax fasciolata). A captive adult female bare-faced curassow presented with lameness, hyporexia, and a non-ulcerative and firm tumor in the right femur. The bird was euthanized due to the poor prognosis. Histopathology revealed an infiltrative mesenchymal neoplasm consisting of spindle cells with moderate cell pleomorphism, organized in bundles and interspersed by marked deposition of the osteoid matrix, which was compatible with osteosarcoma affecting both femur and tibiotarsus, with renal metastasis. Immunohistochemistry of the primary and metastatic tumor demonstrated vimentin expression by neoplastic cells. Samples of the neoplasm, bone marrow, and spleen were processed for PCR, which enabled the demonstration of proviral avian leukosis virus (ALV) DNA. CONCLUSIONS: To the best of our knowledge, this is the first report of an osteosarcoma in a bare-faced curassow with an unusual polyostotic manifestation and associated with ALV infection.
Assuntos
Leucose Aviária , Doenças das Aves/patologia , Neoplasias Ósseas/veterinária , Osteossarcoma/veterinária , Animais , Vírus da Leucose Aviária/isolamento & purificação , Doenças das Aves/virologia , Medula Óssea/virologia , Neoplasias Ósseas/virologia , Feminino , Galliformes/virologia , Neoplasias Renais/secundário , Neoplasias Renais/veterinária , Osteossarcoma/virologia , Baço/virologia , Vimentina/metabolismoRESUMO
BACKGROUND: Hydroa Vacciniforme-like Lymphoproliferative Disorder (HV-LPD) is the name given to a group of Epstein-Barr virus (EBV)-associated diseases. It resembles hydroa vacciniforme (HV), the rarest form of photosensitivity, and is a T-cell disorder associated with an Epstein-Barr virus infection. The majority of diagnosed cases occur in East Asia and South America. It is rare in the United States and Europe. Multiple studies have revealed the clinical manifestation of an enlarged liver, but no gold standard such as pathology has yet supported this as a clinical sign of HV-LPD. CASE PRESENTATION: Here, we report a case of a 34-year-old Asian female with definite liver invasion. The patient had complained of a recurring facial rash for many years. The patient was admitted to the hospital because of an enlarged liver. After hospitalization, she was given an EB virus nucleic acid test. The EB virus nucleic acid test was positive, and pathological examination suggested that HV-LPD had invaded the skin, bone marrow, and liver. After being given antiviral treatment, the patient's symptoms were mitigated. CONCLUSIONS: Our case confirms the liver damage was caused by HV-LPD and the effectiveness of antiviral treatment.
Assuntos
Medula Óssea/patologia , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/diagnóstico , Herpesvirus Humano 4/genética , Hidroa Vaciniforme/complicações , Hidroa Vaciniforme/diagnóstico , Fígado/patologia , Transtornos Linfoproliferativos/complicações , Transtornos Linfoproliferativos/diagnóstico , Adulto , Antivirais/uso terapêutico , Pequim , Medula Óssea/virologia , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Infecções por Vírus Epstein-Barr/virologia , Exantema/complicações , Exantema/tratamento farmacológico , Feminino , Hepatomegalia/tratamento farmacológico , Hepatomegalia/virologia , Humanos , Hidroa Vaciniforme/tratamento farmacológico , Hidroa Vaciniforme/patologia , Fígado/virologia , Linfoma de Células T/complicações , Linfoma de Células T/tratamento farmacológico , Linfoma de Células T/virologia , Transtornos Linfoproliferativos/tratamento farmacológico , Transtornos Linfoproliferativos/patologia , Pele/patologia , Resultado do TratamentoAssuntos
Medula Óssea/patologia , Infecções por Vírus Epstein-Barr/etiologia , Linfonodos/patologia , Transtornos Linfoproliferativos/etiologia , Mielofibrose Primária/tratamento farmacológico , Pirazóis/efeitos adversos , Células de Reed-Sternberg/patologia , Antígenos CD/análise , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Medula Óssea/virologia , DNA Viral/sangue , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Doxorrubicina/administração & dosagem , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Infecções por Vírus Epstein-Barr/patologia , Humanos , Hospedeiro Imunocomprometido , Linfonodos/virologia , Transtornos Linfoproliferativos/tratamento farmacológico , Transtornos Linfoproliferativos/patologia , Masculino , Pessoa de Meia-Idade , Nitrilas , Fator de Transcrição PAX5/análise , Prednisona/administração & dosagem , Mielofibrose Primária/etiologia , Pirimidinas , RNA Viral/análise , Células de Reed-Sternberg/química , Células de Reed-Sternberg/virologia , Trombocitemia Essencial/complicações , Trombocitemia Essencial/genética , Vimblastina/administração & dosagem , GencitabinaRESUMO
AIMS: Haemophagocytosis in the bone marrow of patients who have succumbed to coronavirus disease 19 (COVID-19) has not been widely studied. The aims of the present study were to perform morphological analyses and morphometry of haemophagocytosis in the bone marrow of patients with severe COVID-19, and to correlate the findings with the clinical course of the disease. METHODS AND RESULTS: In this single-centre study performed at the University Hospital Jena, bone marrow specimens of 15 deceased patients who had experienced a severe course of COVID-19 were sampled from the vertebral column during autopsy. Slides of the bone marrow were stained with routine stains or immunohistochemically, and further examined for haemophagocytosis by the use of light microscopy. To substantiate the morphological findings, additional slides were stained for CD163 and morphometry was performed. In all bone marrow samples, an increase in cellularity was found. Haemophagocytes with erythrophagocytosis were detected in 67% of the deceased patients. In tissues with low numbers of haemophagocytes or ill-defined haemophagocytes, an increase in iron deposits was frequently seen. Morphological findings were then correlated with several important clinical data, and the HScore (probability of having a reactive hemophagocytic syndrome) was calculated to posthumously confirm the diagnosis of secondary haemophagocytic lymphohistiocytosis. The median duration of disease and the hospitalisation time were lower in patients with haemophagocytosis (n = 10) than in patients without haemophagocytosis (n = 5). In addition, patients with haemophagocytes showed increased inflammatory parameters 2-5 days prior to death, in contrast to patients without haemophagocytes. CONCLUSIONS: Haemophagocytosis is a common finding in the bone marrow of deceased individuals with severe COVID-19, and may indicate fatal severe acute respiratory syndrome coronavirus 2 infections.
Assuntos
COVID-19/virologia , Linfo-Histiocitose Hemofagocítica/virologia , SARS-CoV-2/fisiologia , Idoso , Idoso de 80 Anos ou mais , Autopsia , Medula Óssea/patologia , Medula Óssea/virologia , COVID-19/complicações , COVID-19/patologia , Feminino , Hospitalização , Humanos , Imuno-Histoquímica , Linfo-Histiocitose Hemofagocítica/complicações , Linfo-Histiocitose Hemofagocítica/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
Europe is currently experiencing a long-lasting African swine fever (ASF) epidemic, both in domestic pigs and wild boar. There is great concern that carcasses of infected wild boar may act as long-term virus reservoirs in the environment. We evaluated the tenacity of ASF virus (ASFV) in tissues and body fluids from experimentally infected domestic pigs and wild boar, which were stored on different matrices and at different temperatures. Samples were analysed at regular intervals for viral genome and infectious virus. ASFV was most stable in spleen or muscles stored at -20 °C and in blood stored at 4 °C. In bones stored at -20 °C, infectious virus was detected for up to three months, and at 4 °C for up to one month, while at room temperature (RT), no infectious virus could be recovered after one week. Skin stored at -20 °C, 4 °C and RT remained infectious for up to three, six and three months, respectively. In urine and faeces, no infectious virus was recovered after one week, irrespective of the matrix. In conclusion, tissues and organs from decomposing carcasses that persist in the environment for a long time can be a source of infection for several months, especially at low temperatures.
Assuntos
Vírus da Febre Suína Africana/isolamento & purificação , Vírus da Febre Suína Africana/fisiologia , Febre Suína Africana/epidemiologia , Sus scrofa/virologia , Vírus da Febre Suína Africana/genética , Animais , Sangue/virologia , Medula Óssea/virologia , Estônia , Fezes/virologia , Genoma Viral , Cinética , Músculos/virologia , Fatores de Risco , Pele/virologia , Baço/virologia , Suínos , Temperatura , Urina/virologiaRESUMO
An effective strategy to cure HIV will likely require a potent and sustained antiviral T cell response. Here we explored the utility of chimeric antigen receptor (CAR) T cells, expressing the CD4 ectodomain to confer specificity for the HIV envelope, to mitigate HIV-induced pathogenesis in bone marrow, liver, thymus (BLT) humanized mice. CAR T cells expressing the 4-1BB/CD3-ζ endodomain were insufficient to prevent viral rebound and CD4+ T cell loss after the discontinuation of antiretroviral therapy. Through iterative improvements to the CAR T cell product, we developed Dual-CAR T cells that simultaneously expressed both 4-1BB/CD3-ζ and CD28/CD3-ζ endodomains. Dual-CAR T cells exhibited expansion kinetics that exceeded 4-1BB-, CD28- and third-generation costimulated CAR T cells, elicited effector functions equivalent to CD28-costimulated CAR T cells and prevented HIV-induced CD4+ T cell loss despite persistent viremia. Moreover, when Dual-CAR T cells were protected from HIV infection through expression of the C34-CXCR4 fusion inhibitor, these cells significantly reduced acute-phase viremia, as well as accelerated HIV suppression in the presence of antiretroviral therapy and reduced tissue viral burden. Collectively, these studies demonstrate the enhanced therapeutic potency of a novel Dual-CAR T cell product with the potential to effectively treat HIV infection.
Assuntos
Antígenos CD4/imunologia , Infecções por HIV/terapia , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos/imunologia , Animais , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Medula Óssea/imunologia , Medula Óssea/virologia , Complexo CD3/antagonistas & inibidores , Antígenos CD4/administração & dosagem , Regulação da Expressão Gênica/imunologia , Proteína gp41 do Envelope de HIV/antagonistas & inibidores , Proteína gp41 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/patogenicidade , Humanos , Fígado/imunologia , Fígado/virologia , Camundongos , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/imunologia , Domínios Proteicos/imunologia , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/imunologia , Receptores de Antígenos Quiméricos/administração & dosagem , Linfócitos T/imunologia , Timo/imunologia , Timo/virologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/antagonistas & inibidoresRESUMO
A critical barrier to the development of a human immunodeficiency virus (HIV) cure is the lack of a scalable animal model that enables robust evaluation of eradication approaches prior to testing in humans. We established a humanized mouse model of latent HIV infection by transplanting "J-Lat" cells, Jurkat cells harboring a latent HIV provirus encoding an enhanced green fluorescent protein (GFP) reporter, into irradiated adult NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ (NSG) mice. J-Lat cells exhibited successful engraftment in several tissues including spleen, bone barrow, peripheral blood, and lung, in line with the diverse natural tissue tropism of HIV. Administration of tumor necrosis factor (TNF)-α, an established HIV latency reversal agent, significantly induced GFP expression in engrafted cells across tissues, reflecting viral reactivation. These data suggest that our murine latency ("µ-Lat") model enables efficient determination of how effectively viral eradication agents, including latency reversal agents, penetrate, and function in diverse anatomical sites harboring HIV in vivo.
Assuntos
Transplante de Células/métodos , Modelos Animais de Doenças , Infecções por HIV/virologia , HIV/fisiologia , Latência Viral , Animais , Medula Óssea/virologia , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , HIV/genética , HIV/patogenicidade , Infecções por HIV/patologia , Infecções por HIV/terapia , Humanos , Células Jurkat , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Provírus/genética , Baço/virologia , Transfecção/métodosRESUMO
Coronavirus disease 2019 (COVID-19) is a global public health emergency with many clinical facets, and new knowledge about its pathogenetic mechanisms is deemed necessary; among these, there are certainly coagulation disorders. In the history of medicine, autopsies and tissue sampling have played a fundamental role in order to understand the pathogenesis of emerging diseases, including infectious ones; compared to the past, histopathology can be now expanded by innovative techniques and modern technologies. For the first time in worldwide literature, we provide a detailed postmortem and biopsy report on the marked increase, up to 1 order of magnitude, of naked megakaryocyte nuclei in the bone marrow and lungs from serious COVID-19 patients. Most likely related to high interleukin-6 serum levels stimulating megakaryocytopoiesis, this phenomenon concurs to explain well the pulmonary abnormal immunothrombosis in these critically ill patients, all without molecular or electron microscopy signs of megakaryocyte infection.
Assuntos
Betacoronavirus/patogenicidade , Medula Óssea/patologia , Infecções por Coronavirus/patologia , Síndrome da Liberação de Citocina/patologia , Coagulação Intravascular Disseminada/patologia , Pulmão/patologia , Pneumonia Viral/patologia , Trombose/patologia , Adulto , Idoso , Autopsia , Betacoronavirus/imunologia , Medula Óssea/imunologia , Medula Óssea/virologia , COVID-19 , Núcleo Celular/imunologia , Núcleo Celular/patologia , Núcleo Celular/virologia , Infecções por Coronavirus/complicações , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Estado Terminal , Síndrome da Liberação de Citocina/complicações , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/virologia , Coagulação Intravascular Disseminada/complicações , Coagulação Intravascular Disseminada/imunologia , Coagulação Intravascular Disseminada/virologia , Evolução Fatal , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interleucina-6/biossíntese , Interleucina-6/imunologia , Pulmão/imunologia , Pulmão/virologia , Masculino , Megacariócitos/imunologia , Megacariócitos/patologia , Megacariócitos/virologia , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , SARS-CoV-2 , Índice de Gravidade de Doença , Trombopoese/imunologia , Trombose/complicações , Trombose/imunologia , Trombose/virologiaRESUMO
While feline leukemia virus (FeLV) has been shown to infect felid species other than the endemic domestic cat host, differences in FeLV susceptibility among species has not been evaluated. Previous reports have noted a negative correlation between endogenous FeLV (enFeLV) copy number and exogenous FeLV (exFeLV) infection outcomes in domestic cats. Since felids outside the genus Felis do not harbor enFeLV genomes, we hypothesized absence of enFeLV results in more severe disease consequences in felid species lacking these genomic elements. We infected primary fibroblasts isolated from domestic cats (Felis catus) and pumas (Puma concolor) with FeLV and quantitated proviral and viral antigen loads. Domestic cat enFeLV env and long terminal repeat (LTR) copy numbers were determined for each individual and compared to FeLV viral outcomes. FeLV proviral and antigen levels were also measured in 6 naturally infected domestic cats and 11 naturally infected Florida panthers (P. concolor coryi). We demonstrated that puma fibroblasts are more permissive to FeLV than domestic cat cells, and domestic cat FeLV restriction was highly related to enFeLV-LTR copy number. Terminal tissues from FeLV-infected Florida panthers and domestic cats had similar exFeLV proviral copy numbers, but Florida panther tissues have higher FeLV antigen loads. Our work indicates that enFeLV-LTR elements negatively correlate with exogenous FeLV replication. Further, Puma concolor samples lacking enFeLV are more permissive to FeLV infection than domestic cat samples, suggesting that endogenization can play a beneficial role in mitigating exogenous retroviral infections. Conversely, presence of endogenous retroelements may relate to new host susceptibility during viral spillover events.IMPORTANCE Feline leukemia virus (FeLV) can infect a variety of felid species. Only the primary domestic cat host and related small cat species harbor a related endogenous virus in their genomes. Previous studies noted a negative association between the endogenous virus copy number and exogenous virus infection in domestic cats. This report shows that puma cells, which lack endogenous FeLV, produce more virus more rapidly than domestic cat fibroblasts following cell culture challenge. We document a strong association between domestic cat cell susceptibility and FeLV long terminal repeat (LTR) copy number, similar to observations in natural FeLV infections. Viral replication does not, however, correlate with FeLV env copy number, suggesting that this effect is specific to FeLV-LTR elements. This discovery indicates a protective capacity of the endogenous virus against the exogenous form, either via direct interference or indirectly via gene regulation, and may suggest evolutionary outcomes of retroviral endogenization.
Assuntos
Variações do Número de Cópias de DNA , Produtos do Gene env/genética , Vírus da Leucemia Felina/genética , Vírus da Leucemia Felina/patogenicidade , Leucemia Felina/virologia , Puma/virologia , Animais , Medula Óssea/patologia , Medula Óssea/virologia , Gatos , Feminino , Fibroblastos/patologia , Fibroblastos/virologia , Produtos do Gene env/metabolismo , Especificidade de Hospedeiro , Vírus da Leucemia Felina/metabolismo , Leucemia Felina/patologia , Linfonodos/patologia , Linfonodos/virologia , Masculino , Cultura Primária de Células , Baço/patologia , Baço/virologia , Sequências Repetidas Terminais , Timo/patologia , Timo/virologia , Carga Viral , Replicação Viral/genéticaAssuntos
Betacoronavirus/imunologia , Medula Óssea/imunologia , Medula Óssea/virologia , Infecções por Coronavirus/imunologia , Hospedeiro Imunocomprometido/imunologia , Pancitopenia/imunologia , Pneumonia Viral/imunologia , Betacoronavirus/metabolismo , Medula Óssea/metabolismo , COVID-19 , Infecções por Coronavirus/sangue , Infecções por Coronavirus/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Pancitopenia/sangue , Pancitopenia/diagnóstico , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/diagnóstico , SARS-CoV-2RESUMO
Pegiviruses frequently cause persistent infection (as defined by >6 months), but unlike most other Flaviviridae members, no apparent clinical disease. Human pegivirus (HPgV, previously GBV-C) is detectable in 1-4% of healthy individuals and another 5-13% are seropositive. Some evidence for infection of bone marrow and spleen exists. Equine pegivirus 1 (EPgV-1) is not linked to disease, whereas another pegivirus, Theiler's disease-associated virus (TDAV), was identified in an outbreak of acute serum hepatitis (Theiler's disease) in horses. Although no subsequent reports link TDAV to disease, any association with hepatitis has not been formally examined. Here, we characterized EPgV-1 and TDAV tropism, sequence diversity, persistence and association with liver disease in horses. Among more than 20 tissue types, we consistently detected high viral loads only in serum, bone marrow and spleen, and viral RNA replication was consistently identified in bone marrow. PBMCs and lymph nodes, but not liver, were sporadically positive. To exclude potential effects of co-infecting agents in experimental infections, we constructed full-length consensus cDNA clones; this was enabled by determination of the complete viral genomes, including a novel TDAV 3' terminus. Clone derived RNA transcripts were used for direct intrasplenic inoculation of healthy horses. This led to productive infection detectable from week 2-3 and persisting beyond the 28 weeks of study. We did not observe any clinical signs of illness or elevation of circulating liver enzymes. The polyprotein consensus sequences did not change, suggesting that both clones were fully functional. To our knowledge, this is the first successful extrahepatic viral RNA launch and the first robust reverse genetics system for a pegivirus. In conclusion, equine pegiviruses are bone marrow tropic, cause persistent infection in horses, and are not associated with hepatitis. Based on these findings, it may be appropriate to rename the group of TDAV and related viruses as EPgV-2.