RESUMO
Elevated levels of miR-155 in solid and liquid malignancies correlate with aggressiveness of the disease. In this manuscript, we show that miR-155 targets transcripts encoding IcosL, the ligand for Inducible T-cell costimulator (Icos), thus impairing the ability of T cells to recognize and eliminate malignant cells. We specifically found that overexpression of miR-155 in B cells of Eµ-miR-155 mice causes loss of IcosL expression as they progress toward malignancy. Similarly, in mice where miR-155 expression is controlled by a Cre-Tet-OFF system, miR-155 induction led to malignant infiltrates lacking IcosL expression. Conversely, turning miR-155 OFF led to tumor regression and emergence of infiltrates composed of IcosL-positive B cells and Icos-positive T cells forming immunological synapses. Therefore, we next engineered malignant cells to express IcosL, in order to determine whether IcosL expression would increase tumor infiltration by cytotoxic T cells and reduce tumor progression. Indeed, overexpressing an IcosL-encoding cDNA in MC38 murine colon cancer cells before injection into syngeneic C57BL6 mice reduced tumor size and increased intratumor CD8+ T cell infiltration, that formed synapses with IcosL-expressing MC38 cells. Our results underscore the fact that by targeting IcosL transcripts, miR-155 impairs the infiltration of tumors by cytotoxic T cells, as well as the importance of IcosL on enhancing the immune response against malignant cells. These findings should lead to the development of more effective anticancer treatments based on maintaining, increasing, or restoring IcosL expression by malignant cells, along with impairing miR-155 activity.
Assuntos
Ligante Coestimulador de Linfócitos T Induzíveis , MicroRNAs , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Camundongos , Ligante Coestimulador de Linfócitos T Induzíveis/metabolismo , Ligante Coestimulador de Linfócitos T Induzíveis/genética , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BL , Humanos , Linfócitos T Citotóxicos/imunologia , Regulação Neoplásica da Expressão Gênica , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Neoplasias/imunologia , Neoplasias/genética , Neoplasias/patologiaRESUMO
Both neurofibrillary tangles and senile plaques are associated with inflammation in Alzheimer's disease (AD). Their relative degree of induced neuroinflammation, however, is not well established. Mouse models of AD that expressed either human Aß42 (n = 7) or human hyperphosphorylated tau protein alone (n = 3), wild type (n = 10), and human AD samples (n = 29 with 18 controls) were studied. The benefit of using mouse models that possess only human tau or amyloid-b is that it allows for the individual evaluation of how each protein affects neuroinflammation, something not possible in human tissue. Three indicators of neuroinflammation were examined: TLRs/RIG1 expression, the density of astrocytes and microglial cells, and well-established mediators of neuroinflammation (IL6, TNFα, IL1ß, and CXCL10). There was a statistically significant increase in neuroinflammation with all three variables in the mouse models with human tau only as compared to human Aß42 only or wild-type mice (each at p < 0.0001). Only the Aß42 5xFAD mice (n = 4) showed statistically higher neuroinflammation versus wild type (p = 0.0030). The human AD tissues were segregated into Aß42 only or hyperphosphorylated tau protein with Aß42. The latter areas showed increased neuroinflammation with each of the three variables compared to the areas with only Aß42. Of the TLRs and RIG-1, TLR8 was significantly elevated in both the mouse model and human AD and only in areas with the abnormal tau protein. It is concluded that although Aß42 and hyperphosphorylated tau protein can each induce inflammation, the latter protein is associated with a much stronger neuroinflammatory response vis-a-vis a significantly greater activated microglial response.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Camundongos Transgênicos , Doenças Neuroinflamatórias , Proteínas tau , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Humanos , Peptídeos beta-Amiloides/metabolismo , Fosforilação , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , Feminino , Masculino , Fragmentos de Peptídeos/metabolismo , Camundongos , Inflamação/patologia , Inflamação/metabolismo , Idoso , Microglia/metabolismo , Microglia/patologia , Modelos Animais de Doenças , Idoso de 80 Anos ou mais , Astrócitos/metabolismo , Astrócitos/patologiaRESUMO
PURPOSE: Our preclinical studies showed that the oncolytic reovirus formulation pelareorep (PELA) has significant immunomodulatory anti-myeloma activity. We conducted an investigator-initiated clinical trial to evaluate PELA in combination with dexamethasone (Dex) and bortezomib (BZ) and define the tumor immune microenvironment (TiME) in patients with multiple myeloma treated with this regimen. PATIENTS AND METHODS: Patients with relapsed/refractory multiple myeloma (n = 14) were enrolled in a phase Ib clinical trial (ClinicalTrials.gov: NCT02514382) of three escalating PELA doses administered on Days 1, 2, 8, 9, 15, and 16. Patients received 40 mg Dex and 1.5 mg/m2 BZ on Days 1, 8, and 15. Cycles were repeated every 28 days. Pre- and posttreatment bone marrow specimens (IHC, n = 9; imaging mass cytometry, n = 6) and peripheral blood samples were collected for analysis (flow cytometry, n = 5; T-cell receptor clonality, n = 7; cytokine assay, n = 7). RESULTS: PELA/BZ/Dex was well-tolerated in all patients. Treatment-emergent toxicities were transient, and no dose-limiting toxicities occurred. Six (55%) of 11 response-evaluable patients showed decreased paraprotein. Treatment increased T and natural killer cell activation, inflammatory cytokine release, and programmed death-ligand 1 expression in bone marrow. Compared with nonresponders, responders had higher reovirus protein levels, increased cytotoxic T-cell infiltration posttreatment, cytotoxic T cells in significantly closer proximity to multiple myeloma cells, and larger populations of a novel immune-primed multiple myeloma phenotype (CD138+ IDO1+HLA-ABCHigh), indicating immunomodulation. CONCLUSIONS: PELA/BZ/Dex is well-tolerated and associated with anti-multiple myeloma activity in a subset of responding patients, characterized by immune reprogramming and TiME changes, warranting further investigation of PELA as an immunomodulator.
Assuntos
Mieloma Múltiplo , Terapia Viral Oncolítica , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/etiologia , Terapia Viral Oncolítica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Bortezomib/uso terapêutico , Dexametasona/uso terapêutico , Citocinas/uso terapêutico , Microambiente TumoralRESUMO
Persistent HPV16 infection is a major cause of the global cancer burden. The viral life cycle is dependent on the differentiation program of stratified squamous epithelium, but the landscape of keratinocyte subpopulations which support distinct phases of the viral life cycle has yet to be elucidated. Here, single cell RNA sequencing of HPV16 infected compared to uninfected organoids identifies twelve distinct keratinocyte populations, with a subset mapped to reconstruct their respective 3D geography in stratified squamous epithelium. Instead of conventional terminally differentiated cells, an HPV-reprogrammed keratinocyte subpopulation (HIDDEN cells) forms the surface compartment and requires overexpression of the ELF3/ESE-1 transcription factor. HIDDEN cells are detected throughout stages of human carcinogenesis including primary human cervical intraepithelial neoplasias and HPV positive head and neck cancers, and a possible role in promoting viral carcinogenesis is supported by TCGA analyses. Single cell transcriptome information on HPV-infected versus uninfected epithelium will enable broader studies of the role of individual keratinocyte subpopulations in tumor virus infection and cancer evolution.
Assuntos
Carcinoma de Células Escamosas , Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Feminino , Humanos , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Transcriptoma , Epitélio/metabolismo , Queratinócitos/metabolismo , Carcinogênese/genética , Carcinoma de Células Escamosas/genética , Proteínas Oncogênicas Virais/genéticaRESUMO
The microbiological etiology of seasonal upper respiratory illnesses in the United States is dominated by viruses, including influenza A, B, respiratory syncytial virus, and SARS-CoV2. Mycoplasma pneumonia, treatable with antibiotics, can also cause upper respiratory symptoms and is typically associated with about 15 % of cases. There is no clinical or radiologic finding diagnostic of Mycoplasma pneumonia infection and PCR-based testing is not routinely used in the clinical setting. Further, the bacteria grows slowly in culture and the diagnostic IgM response will take days after the onset of infection. Thus, a rapid diagnostic test for Mycobacterium pneumonia infection is needed. This study documented two cases of Mycoplasma pneumonia infection of the upper respiratory system using in situ hybridization in a series of over 20 patients who were being tested for SARS-CoV2 infection. The respiratory secretions were placed on a glass slide, fixed in 10 % buffered formalin, and then tested using a Mycoplasma pneumonia probe. The high bacterial number associated with acute infection allowed for straightforward detection by in situ hybridization in a few hours. Antibiotic therapy led to rapid resolution of the symptoms. This highlights the ability of standard in situ hybridization as a rapid diagnostic test for Mycoplasma pneumonia in the clinical setting.
Assuntos
COVID-19 , Pneumonia por Mycoplasma , Humanos , Pneumonia por Mycoplasma/diagnóstico , Pneumonia por Mycoplasma/tratamento farmacológico , Pneumonia por Mycoplasma/microbiologia , RNA Viral , SARS-CoV-2 , Hibridização In Situ , Teste para COVID-19RESUMO
Lichen sclerosus (LS) is a cutaneous disease of unknown etiology that often involves the vulva or foreskin but also can affect extragenital sites. Regardless of the anatomic site, the histomorphology and presumably pathogenesis are similar. Perhaps a clue to the pathophysiology of LS lies in its frequent association with morphea, specifically, when occurring in an extragenital context. In our experience a striking feature evident in established lichen sclerosis (LS) is one of superficial vascular drop out whereby residual vessels exhibited endothelial cell necrosis and microvascular basement membrane zone thickening, the latter reflective of antecedent episodes of microvascular injury. We sought to understand the pathophysiology that underlies the distinct vascular changes and in doing so, shed light on the pathogenesis of LS. We examined 44 cases of LS over a period of 2019 to 2021. We were able to obtain past medical histories in 34 of the 44 cases. Regarding pathological assessment, the predominant focus was on microvascular changes. We assessed the role of C5b-9 mediated vascular injury in the pathogenesis of the vasculopathy and enhanced type I interferon signaling in vessels given the morphologic semblance to the select interferonopathy syndromes, namely fibrosing dermatomyositis and Kohlmeier Degos disease. We examined the expression of CMV DNA and protein based on prior observations in an earlier study that isolated early protein expression in the microvasculature in the setting of LS and scleroderma. From a clinical perspective, the most striking association was an older age at the time of diagnosis (mean age of 62 years and median age of 61.5 years) and the presence of vascular comorbidities of diabetes, hypertension, and hyperlipidemia in almost 80% of cases. All cases showed significant microvascular changes in the superficial corium with the most frequent findings being those of significant basement membrane zone reduplication and vascular drop out. A number of cases showed prominent microvascular deposits of C5b-9 in the zone of hyalinizing fibrosis or subjacent to the discernible table of fibroplasia in the absence of enhanced type I interferon signaling. In no case were there viral cytopathic changes associated with CMV affecting the endothelium. The studies that encode CMV DNA or protein did not show a significant role for CMV reactivation in endothelium in the majority of the studied cases. It is concluded that the pathophysiology of LS includes a microvascular injury syndrome within the papillary dermis. The mechanism of endothelial cell injury is complement mediated at least in part and could reflect an adaptive immune response targeting endothelium indicative of classic complement pathway activation when coexisting with morphea or occurring in younger individuals. A non-immune based endothelial dysfunction and complement mediated injury unrelated to antibody driven classic complement pathway activation are more likely pathogenetically in the setting of certain diseases like diabetes mellitus and hypertension. Vascular drop out can be explained by the diminished endothelial progenitor pool needed to repopulate the damaged microvessels in certain settings like hypertension and diabetes.
Assuntos
Infecções por Citomegalovirus , Hipertensão , Interferon Tipo I , Líquen Escleroso e Atrófico , Esclerodermia Localizada , Adulto , Humanos , Pessoa de Meia-Idade , Complexo de Ataque à Membrana do Sistema Complemento , Infecções por Citomegalovirus/complicações , Hipertensão/complicações , Líquen Escleroso e Atrófico/complicações , Líquen Escleroso e Atrófico/patologia , Esclerodermia Localizada/complicações , Esclerodermia Localizada/patologiaRESUMO
The toll like receptors (TLRs) and RIG-1 are proteins involved in the initial reaction of the innate immune system to infectious diseases and, thus, can provide much information to the surgical pathologist in terms of the molecular dynamics of the infection. The TLRs (TLR1, 2, 3, 4, 7, 8) and RIG-1 distribution as determined by immunohistochemistry was examined in the following diseases: human papillomavirus (n = 30 including 15 squamous intraepithelial lesions (SIL), 5 cancers, and 10 controls); molluscum contagiosum (n = 8 including 4 controls), SARS-CoV2 (n = 52 including 20 mild, 5 fatal, and 27 controls) and reovirus infection as oncolytic therapy. Mild, regressing infection (molluscum contagiosum, mild SARS-CoV2 and low grade SIL) each showed the same pattern: marked up regulation of at least three of the TLRs/RIG-1 with decreased expression of none compared to the controls. Severe infection (fatal SARS-CoV2, and cervical cancer) each showed marked decrease expression in at least three of the TLRs/RIG-1. We recently documented an equivalent marked decrease expression of the TLRs/RIG-1 in the placenta in fatal in utero infections. The reoviral infected tissues showed an overall pattern of marked increase expression of TLRs/RIG-1, consistent with a strong anti-viral response. Thus, the in situ testing of infectious diseases by a panel of these early infectious disease recognition proteins may allow the surgical pathologist to predict the outcome of the disease which, in turn, may assist in the understanding of the role of the TLRs/RIG-1 in determining the fate of a given infectious process.
Assuntos
Doenças Transmissíveis , Proteína DEAD-box 58 , Receptores Toll-Like , Feminino , Humanos , Gravidez , Doenças Transmissíveis/genética , Doenças Transmissíveis/patologia , COVID-19/genética , COVID-19/patologia , Molusco Contagioso/genética , Molusco Contagioso/patologia , RNA Viral , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Receptores Toll-Like/metabolismo , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismoRESUMO
Novel biomarkers of in utero infections are needed to help guide early therapy. The toll like receptors (TLRs) and retinoic acid-inducible gene 1 (RIG-1) are proteins involved in the initial reaction of the innate immune system to infectious diseases. This study tested the hypothesis that a panel of TLRs and RIG-1 in the placenta could serve as an early biomarker of in utero infections. The TLRs and RIG-1 expression as determined by immunohistochemistry was scored in 10 control placentas (normal delivery or neonatal damage from known non-infectious cause), 8 placentas from documented in utero bacterial infection, and 7 placentas from documented in utero viral infections blinded to the clinical information. The non-infected placentas showed the following profile: no expression (TLR1, TLR3, TLR4, TLR7, TLR8), moderate expression (TLR2), and strong expression (RIG-1). The bacterial and viral infection cases shared the following profile: no to mild expression (TLR 2, TLR7, and RIG1), moderate expression (TLR4), and strong expression (TLR1, TLR3, and TLR8). The histologic findings in the chorionic villi were equivalent in the infected cases and controls, underscoring the need for molecular testing by the surgical pathologist when in utero infection is suspected. The results suggest that a panel of TLRs/RIG-1 analyses can allow the pathologist and/or clinician to diagnose in utero infections soon after birth. Also, treatments to antagonize the effects of TLR1, 3, and 8 may help abrogate in utero neonatal damage.
Assuntos
Placenta , Complicações Infecciosas na Gravidez , Feminino , Humanos , Recém-Nascido , Gravidez/imunologia , Placenta/imunologia , Placenta/metabolismo , Receptor 1 Toll-Like/genética , Receptor 1 Toll-Like/metabolismo , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like , Receptor 7 Toll-Like , Receptor 8 Toll-Like/genética , Receptor 8 Toll-Like/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , Complicações Infecciosas na Gravidez/genética , Complicações Infecciosas na Gravidez/metabolismoRESUMO
Pre-existing Alzheimer's disease is a risk factor for severe/fatal COVID-19 and infection by SARS-CoV2 virus has been associated with an increased incidence of un-masked Alzheimer's disease. The molecular basis whereby SARS-CoV2 may amplify Alzheimer's disease is not well understood. This study analyzed the molecular changes in autopsy brain tissues from people with pre-existing dementia who died of COVID-19 (n = 5) which was compared to equivalent tissues of people who died of COVID-19 with no history of dementia (n = 8), Alzheimer's disease pre-COVID-19 (n = 10) and aged matched controls (n = 10) in a blinded fashion. Immunohistochemistry analyses for hyperphosphorylated tau protein, α-synuclein, and ß-amyloid-42 confirmed the diagnoses of Alzheimer's disease (n = 4), and Lewy body dementia (n = 1) in the COVID-19 group. The brain tissues from patients who died of COVID-19 with no history of dementia showed a diffuse microangiopathy marked by endocytosis of spike subunit S1 and S2 in primarily CD31+ endothelia with strong co-localization with ACE2, Caspase-3, IL6, TNFα, and Complement component 6 that was not associated with SARS-CoV2 RNA. Microglial activation marked by increased TMEM119 and MCP1 protein expression closely paralleled the endocytosed spike protein. The COVID-19 tissues from people with no pre-existing dementia showed, compared to controls, 5-10× fold increases in expression of neuronal NOS and NMDAR2 as well as a marked decrease in the expression of proteins whose loss is associated with worsening Alzheimer's disease: MFSD2a, SHIP1, BCL6, BCL10, and BACH1. In COVID-19 tissues from people with dementia the widespread spike-induced microencephalitis with the concomitant microglial activation co-existed in the same areas where neurons had hyperphosphorylated tau protein suggesting that the already dysfunctional neurons were additionally stressed by the SARS-CoV2 induced microangiopathy. ACE2+ human brain endothelial cells treated with high dose (but not vaccine equivalent low dose) spike S1 protein demonstrated each of the molecular changes noted in the in vivo COVID-19 and COVID-19/Alzheimer's disease brain tissues. It is concluded that fatal COVID-19 induces a diffuse microencephalitis and microglial activation in the brain due to endocytosis of circulating viral spike protein that amplifies pre-existing dementia in at least two ways: 1) modulates the expression of proteins that may worsen Alzheimer's disease and 2) stresses the already dysfunctional neurons by causing an acute proinflammatory/hypercoagulable/hypoxic microenvironment in areas with abundant hyperphosphorylated tau protein and/or ßA-42.
Assuntos
Doença de Alzheimer , COVID-19 , Idoso , Humanos , Doença de Alzheimer/complicações , Doença de Alzheimer/genética , Enzima de Conversão de Angiotensina 2 , COVID-19/complicações , Células Endoteliais/metabolismo , RNA Viral , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Proteínas tau/metabolismo , Sistema Nervoso CentralRESUMO
This study compared the immune response in mild versus fatal SARS-CoV2 infection. Forty nasopharyngeal swabs with either productive mild infection (n = 20) or negative for SARS-CoV2 (n = 20) were tested along with ten lung sections from people who died of COVID-19 which contained abundant SARS-CoV2 and ten controls. There was a 25-fold increase in the CD3+T cell numbers in the viral positive nasopharyngeal swabs compared to the controls (p < 0.001) and no change in the CD3+T cell count in the fatal COVID-19 lungs versus the controls. CD11b + and CD206+ macrophage counts were significantly higher in the mild versus fatal disease (p = 0.002). In situ analysis for SARS-CoV2 RNA found ten COVID-19 lung sections that had no/rare detectable virus and also lacked the microangiopathy typical of the viral positive sections. These viral negative lung tissues when compared to the viral positive lung samples showed a highly significant increase in CD3+ and CD8 T cells (p < 0.001), equivalent numbers of CD163+ cells, and significantly less PDL1, CD11b and CD206+ cells (p = 0.002). It is concluded that mild SARS-CoV2 infection is marked by a much stronger CD3/CD8 T cell, CD11b, and CD206 macrophage response than the fatal lung disease where viral RNA is abundant.
Assuntos
COVID-19 , Pneumonia Viral , Humanos , RNA Viral , SARS-CoV-2 , ImunidadeRESUMO
ABSTRACT: Human papilloma virus (HPV) is the causative agent for a variety of cutaneous lesions including verruca vulgaris (VV) and epidermodysplasia verruciformis (EDV). There are more than 200 known genotypes of HPV, and specific HPV types are associated with different clinical manifestations and malignant potentials. Herein, we describe a case of a 43-year-old immunocompetent woman who presented with morphologically distinct lesions that were most consistent with EDV on clinical examination. However, further histopathological and viral analysis confirmed the lesions as HPV-57-positive VV. The risk of malignant transformation, and therefore treatment and surveillance, is dramatically different in VV versus EDV. Therefore, this case highlights the importance of a proper histopathological diagnosis with HPV viral testing when clinical presentations may vary or mimic other diseases.
Assuntos
Alphapapillomavirus , Epidermodisplasia Verruciforme , Infecções por Papillomavirus , Verrugas , Adulto , Alphapapillomavirus/genética , DNA Viral , Epidermodisplasia Verruciforme/patologia , Feminino , Humanos , Papillomaviridae/genética , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/patologiaRESUMO
Cardiac manifestations are common in severe COVID-19. This study compared the histologic, viral, and molecular findings in cardiac tissue in fatal COVID-19 (n = 11) and controls (n = 11). In situ hybridization (SARS-CoV2 RNA) and immunohistochemistry for viral proteins and the host response were quantified for the samples and compared with qRTPCR and Western blot data. Control hearts showed a high resident population of macrophages that had variable ACE2 expression. Cardiac ACE2 expression was 10× greater in the heart tissues of cases and controls with obesity or type II diabetes. Multifocal endothelial cell swelling and degeneration, perivascular edema plus microvascular thrombi were unique to the cases. SARS-CoV2 RNA and nucleocapsid protein were rarely detected in situ in any COVID-19 heart. However, in each case abundant SARS-CoV-2 spike protein was evident. Co-expression experiments showed that the spike protein localized mostly to the ACE2+ interstitial macrophages/pericytes that were activated as evidenced by increased IL6 and TNFα expression. Western blots confirmed the presence of the viral spike protein, but not the nucleocapsid protein, in the cardiac homogenates. The intercalated disc proteins connexin 43, the primary cardiac gap junction protein, and NaV1.5, the predominant cardiac sodium channel, each showed marked lateral migration in the myocytes in the cases, which would increase the risk of reentrant arrhythmias. It is concluded that the viral spike protein, endocytosed by macrophages/pericytes, can induce a myocarditis with the possibility of conduction dysfunction due to abnormal localization of key intercalated disc proteins.