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1.
Proc Natl Acad Sci U S A ; 114(38): 10202-10207, 2017 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-28874546

RESUMO

The earlier diagnosis of cancer is one of the keys to reducing cancer deaths in the future. Here we describe our efforts to develop a noninvasive blood test for the detection of pancreatic ductal adenocarcinoma. We combined blood tests for KRAS gene mutations with carefully thresholded protein biomarkers to determine whether the combination of these markers was superior to any single marker. The cohort tested included 221 patients with resectable pancreatic ductal adenocarcinomas and 182 control patients without known cancer. KRAS mutations were detected in the plasma of 66 patients (30%), and every mutation found in the plasma was identical to that subsequently found in the patient's primary tumor (100% concordance). The use of KRAS in conjunction with four thresholded protein biomarkers increased the sensitivity to 64%. Only one of the 182 plasma samples from the control cohort was positive for any of the DNA or protein biomarkers (99.5% specificity). This combinatorial approach may prove useful for the earlier detection of many cancer types.


Assuntos
Antígeno CA-19-9/sangue , Carcinoma Ductal Pancreático/diagnóstico , DNA Tumoral Circulante/sangue , Neoplasias Pancreáticas/diagnóstico , Proteínas Proto-Oncogênicas p21(ras)/genética , Idoso , Carcinoma Ductal Pancreático/sangue , Carcinoma Ductal Pancreático/genética , Estudos de Casos e Controles , Feminino , Genes p53 , Humanos , Biópsia Líquida , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/genética
2.
PLoS Pathog ; 12(12): e1006104, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27959961

RESUMO

Defining the correlates of immune protection conferred by SIVΔnef, the most effective vaccine against SIV challenge, could enable the design of a protective vaccine against HIV infection. Here we provide a comprehensive assessment of immune responses that protect against SIV infection through detailed analyses of cellular and humoral immune responses in the blood and tissues of rhesus macaques vaccinated with SIVΔnef and then vaginally challenged with wild-type SIV. Despite the presence of robust cellular immune responses, animals at 5 weeks after vaccination displayed only transient viral suppression of challenge virus, whereas all macaques challenged at weeks 20 and 40 post-SIVΔnef vaccination were protected, as defined by either apparent sterile protection or significant suppression of viremia in infected animals. Multiple parameters of CD8 T cell function temporally correlated with maturation of protection, including polyfunctionality, phenotypic differentiation, and redistribution to gut and lymphoid tissues. Importantly, we also demonstrate the induction of a tissue-resident memory population of SIV-specific CD8 T cells in the vaginal mucosa, which was dependent on ongoing low-level antigenic stimulation. Moreover, we show that vaginal and serum antibody titers inversely correlated with post-challenge peak viral load, and we correlate the accumulation and affinity maturation of the antibody response to the duration of the vaccination period as well as to the SIVΔnef antigenic load. In conclusion, maturation of SIVΔnef-induced CD8 T cell and antibody responses, both propelled by viral persistence in the gut mucosa and secondary lymphoid tissues, results in protective immune responses that are able to interrupt viral transmission at mucosal portals of entry as well as potential sites of viral dissemination.


Assuntos
Anticorpos Antivirais/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Feminino , Citometria de Fluxo , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Macaca mulatta , Reação em Cadeia da Polimerase em Tempo Real , Vagina/imunologia , Vagina/virologia , Carga Viral
3.
PLoS Pathog ; 11(2): e1004633, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25688559

RESUMO

The live attenuated simian immunodeficiency virus (LASIV) vaccine SIVΔnef is one of the most effective vaccines in inducing protection against wild-type lentiviral challenge, yet little is known about the mechanisms underlying its remarkable protective efficacy. Here, we exploit deep sequencing technology and comprehensive CD8 T cell epitope mapping to deconstruct the CD8 T cell response, to identify the regions of immune pressure and viral escape, and to delineate the effect of epitope escape on the evolution of the CD8 T cell response in SIVΔnef-vaccinated animals. We demonstrate that the initial CD8 T cell response in the acute phase of SIVΔnef infection is mounted predominantly against more variable epitopes, followed by widespread sequence evolution and viral escape. Furthermore, we show that epitope escape expands the CD8 T cell repertoire that targets highly conserved epitopes, defined as anentropic specificity, and generates de novo responses to the escaped epitope variants during the vaccination period. These results correlate SIVΔnef-induced protection with expanded anentropic specificity and increased response depth. Importantly, these findings render SIVΔnef, long the gold standard in HIV/SIV vaccine research, as a proof-of-concept vaccine that highlights the significance of the twin principles of anentropic specificity and repertoire depth in successful vaccine design.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Evasão da Resposta Imune/imunologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Citometria de Fluxo , Macaca mulatta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Síndrome de Imunodeficiência Adquirida dos Símios/terapia , Vacinas Atenuadas/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologia
4.
Blood ; 118(12): 3321-30, 2011 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-21791421

RESUMO

Natural killer (NK) cells are classically viewed as effector cells that kill virus-infected and neoplastic cells, but recent studies have identified a rare mucosal NK- cell subpopulation secreting the TH17 cytokine IL-22. Here, we report identification of 2 distinct lineages of mucosal NK cells characterized as NKG2A(+)NFIL3(+)RORC(-) and NKp44(+)NFIL3(+)RORC(+). NKG2A(+) NK cells were systemically distributed, cytotoxic, and secreted IFN-γ, whereas NKp44(+) NK cells were mucosae-restricted, noncytotoxic, and produced IL-22 and IL-17. During SIV infection, NKp44(+) NK cells became apoptotic, were depleted, and had an altered functional profile characterized by decreased IL-17 secretion; increased IFN-γ secretion; and, surprisingly, increased potential for cytotoxicity. NKp44(+) NK cells showed no evidence of direct SIV infection; rather, depletion and altered function were associated with SIV-induced up-regulation of inflammatory mediators in the gut, including indoleamine 2,3-dioxygenase 1. Furthermore, treatment of NKp44(+) NK cells with indoleamine 2,3-dioxygenase 1 catabolites in vitro ablated IL-17 production in a dose-dependent manner, whereas other NK-cell functions were unaffected. Thus lentiviral infection both depletes and modifies the functional repertoire of mucosal NK cells involved in the maintenance of gut integrity, a finding that highlights the plasticity of this rare mucosal NK-cell population.


Assuntos
Citotoxicidade Imunológica/efeitos dos fármacos , Mucosa Gástrica/efeitos dos fármacos , Imunidade nas Mucosas/efeitos dos fármacos , Indolamina-Pirrol 2,3,-Dioxigenase/farmacologia , Inflamação/imunologia , Interleucina-17/antagonistas & inibidores , Células Matadoras Naturais/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Linhagem da Célula/imunologia , Citotoxicidade Imunológica/imunologia , Citometria de Fluxo , Mucosa Gástrica/imunologia , Mucosa Gástrica/metabolismo , Imunidade nas Mucosas/imunologia , Inflamação/metabolismo , Inflamação/patologia , Inflamação/virologia , Interferon gama/biossíntese , Interleucina-17/biossíntese , Interleucinas/biossíntese , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Macaca mulatta , Receptor 2 Desencadeador da Citotoxicidade Natural/imunologia , Cultura Primária de Células , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/crescimento & desenvolvimento , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Interleucina 22
5.
J Infect Dis ; 206(9): 1462-8, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22711907

RESUMO

Multiple studies suggest that plasmacytoid dendritic cells (pDCs) are depleted and dysfunctional during human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) infection, but little is known about pDCs in the gut-the primary site of virus replication. Here, we show that during SIV infection, pDCs were reduced 3--fold in the circulation and significantly upregulated the gut-homing marker α4ß7, but were increased 4-fold in rectal biopsies of infected compared to naive macaques. These data revise the understanding of pDC immunobiology during SIV infection, indicating that pDCs are not necessarily depleted, but instead may traffic to and accumulate in the gut mucosa.


Assuntos
Células Dendríticas/imunologia , Trato Gastrointestinal/imunologia , Mucosa Intestinal/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Biópsia , Citometria de Fluxo , Trato Gastrointestinal/patologia , Expressão Gênica , Imuno-Histoquímica , Integrinas/biossíntese , Mucosa Intestinal/patologia , Macaca mulatta , Microscopia de Fluorescência , Reto/imunologia , Reto/patologia , Síndrome de Imunodeficiência Adquirida dos Símios/patologia
6.
Blood ; 115(22): 4439-46, 2010 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-20339088

RESUMO

Natural killer (NK) cells contribute to control of HIV/SIV infection. We defined macaque NK-cell subsets based on expression of CD56 and CD16 and found their distribution to be highly disparate. CD16(+) NK cells predominated in peripheral blood, whereas most mucosal NK cells were CD56(+), and lymph nodes contained both CD56(+) and CD16(-)CD56(-) (double-negative [DN]) subsets. Functional profiles were also distinct among subsets--CD16(+) NK cells expressed high levels of cytolytic molecules, and CD56(+) NK cells were predominantly cytokine-secreting cells, whereas DN NK possessed both functions. In macaques chronically infected with SIV, circulating CD16(+) and DN NK cells were expanded in number and, although markers of cytoxicity increased, cytokine secretion decreased. Notably, CD56(+) NK cells in SIV-infected animals up-regulated perforin, granzyme B, and CD107a. In contrast, the lymph node-homing molecules CD62 ligand (CD62L) and C-C chemokine receptor type 7 (CCR7), which are expressed primarily on CD56(+) and DN NK cells, were significantly down-regulated on NK cells from infected animals. These data demonstrate that SIV infection drives a shift in NK-cell function characterized by decreased cytokine production, expanded cytotoxicity, and trafficking away from secondary lymphoid organs, suggesting that the NK-cell repertoire is not only heterogeneous but also plastic.


Assuntos
Células Matadoras Naturais/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Animais , Antígeno CD56/metabolismo , Degranulação Celular , Citocinas/biossíntese , Citotoxicidade Imunológica , Feminino , Granzimas/metabolismo , Células Matadoras Naturais/classificação , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Tecido Linfoide/patologia , Macaca mulatta , Masculino , Mucosa/imunologia , Mucosa/metabolismo , Mucosa/patologia , Perforina/metabolismo , Receptores de IgG/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/patologia
7.
Am Ann Deaf ; 166(4): 527-553, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35185037

RESUMO

The study investigated the literacy-learning conditions of a group of deaf and hard of hearing (d/Dhh) and hearing preschoolers in a mainstream kindergarten sign bilingualism and coenrollment (SLCO) program. The data came from the children's scores on tests of Chinese vocabulary and written Chinese grammar, and questionnaire responses on literacy-learning conditions at home (from parents) and in school (from teachers). The d/Dhh children's performance on the two tests, when compared with that of their hearing peers, suggested that adding sign language and Deaf teachers to the SLCO classroom did not adversely affect the d/Dhh children's literacy learning. Responses to the two questionnaires indicated that parents' and teachers' efforts to organize literacy resources and activities interacted with the children's vocabulary development. These preliminary results encourage more research to elucidate further the relationship between environmental factors and d/Dhh children's literacy development.


Assuntos
Surdez , Alfabetização , Criança , Audição , Hong Kong , Humanos , Desenvolvimento da Linguagem , Vocabulário
8.
Science ; 359(6378): 926-930, 2018 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-29348365

RESUMO

Earlier detection is key to reducing cancer deaths. Here, we describe a blood test that can detect eight common cancer types through assessment of the levels of circulating proteins and mutations in cell-free DNA. We applied this test, called CancerSEEK, to 1005 patients with nonmetastatic, clinically detected cancers of the ovary, liver, stomach, pancreas, esophagus, colorectum, lung, or breast. CancerSEEK tests were positive in a median of 70% of the eight cancer types. The sensitivities ranged from 69 to 98% for the detection of five cancer types (ovary, liver, stomach, pancreas, and esophagus) for which there are no screening tests available for average-risk individuals. The specificity of CancerSEEK was greater than 99%: only 7 of 812 healthy controls scored positive. In addition, CancerSEEK localized the cancer to a small number of anatomic sites in a median of 83% of the patients.


Assuntos
DNA Tumoral Circulante/genética , Detecção Precoce de Câncer/métodos , Testes Hematológicos , Proteínas de Neoplasias/sangue , Neoplasias/diagnóstico , Neoplasias/cirurgia , Custos e Análise de Custo , Detecção Precoce de Câncer/economia , Testes Hematológicos/economia , Humanos , Mutação , Neoplasias/sangue , Neoplasias/genética , Reação em Cadeia da Polimerase/métodos
9.
Hum Gene Ther ; 18(7): 653-64, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17600461

RESUMO

In light of findings demonstrating that the macaque TRIM5alpha protein inhibits infection of cells by human immunodeficiency virus (HIV)-1, simian immunodeficiency virus (SIV)-based lentiviral vectors may have distinct advantages over HIV-1 vectors for the transduction of macaque hematopoietic stem cells. We evaluated the ability of an SIV vector (VRX859) encoding an antisense SIV envelope sequence and enhanced green fluorescent protein (GFP) to inhibit viral replication and to transduce rhesus CD34(+) lymphoid progenitor cells. After infection with homologous SIV strains, CD4(+) cell lines transduced with VRX859 exhibited more than 600-fold inhibition of viral replication compared with control cells. Less inhibition was observed with the divergent SIV strain SIVsmE660. Partial inhibition of a chimeric simian-human immunodeficiency virus, which contains an HIV-1 envelope in an SIV backbone, was observed, suggesting that the SIV vector also contributes to viral inhibition independent of the antisense envelope inhibitor. Transduction of rhesus CD34(+) cells with VRX859 at various multiplicities of infection resulted in transduction efficiencies comparable to those obtained with the HIV vector VRX494. However, when we evaluated transduction of rhesus T lymphocyte progenitors by examining GFP expression in CD4(+) T cells derived from transduced CD34(+) cells, we observed more efficient transduction with the SIV-based vector. GFP(+)CD4(+) T cells derived from VRX859-transduced CD34(+) cells strongly inhibited SIVmac239 replication as compared with control CD4(+) T cells. The ability of this SIV-based vector to mediate potent inhibition of SIV replication, coupled with its efficient transduction of rhesus hematopoietic progenitor cells, make it an important candidate for proof-of-principle experiments of stem cell gene therapy in the SIV-macaque model.


Assuntos
Genes env , Vetores Genéticos , Lentivirus/genética , Oligonucleotídeos Antissenso/farmacologia , Vírus da Imunodeficiência Símia/genética , Replicação Viral/efeitos dos fármacos , Animais , Antígenos CD34/metabolismo , Linhagem Celular , Células Cultivadas , Citometria de Fluxo , Corantes Fluorescentes/metabolismo , Genes env/genética , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Linfócitos T/metabolismo , Transdução Genética
10.
AIDS Res Ther ; 4: 24, 2007 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-17941994

RESUMO

BACKGROUND: Internal polymerase III promoters in retroviral vectors have been used extensively to express short RNA sequences, such as ribozymes, RNA aptamers or short interfering RNA inhibitors, in various positions and orientations. However, the stability of these promoters in the reverse orientation has not been rigorously evaluated. RESULTS: A series of retroviral vectors was generated carrying the U6+1 promoter with 3 different HIV-1 RT-specific RNA aptamers and one control aptamer, all in the reverse orientation. After shuttle packaging, the CD4+ cell line CEMx174 was transduced with each vector, selected for expression of GFP, and challenged with HIV-1. We did not observe inhibition of HIV-1 replication in these transduced populations. PCR amplification of the U6+1 promoter-RNA aptamer inhibitor cassette from transduced CEMx174 cells and RT-PCR amplification from transfected Phoenix (amphotropic) packaging cells showed two distinct products: a full-length product of the expected size as well as a truncated product. The sequence of the full-length PCR product was identical to the predicted amplicon sequence. However, sequencing of the truncated product revealed a 139 bp deletion in the U6 promoter. This deletion decreased transcriptional activity of the U6 promoter. Analysis of the deleted sequences from the U6 promoter in the antisense direction indicated consensus splice donor, splice acceptor and branch point sequences. CONCLUSION: The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.

11.
Nat Genet ; 49(3): 358-366, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28092682

RESUMO

The extent of heterogeneity among driver gene mutations present in naturally occurring metastases-that is, treatment-naive metastatic disease-is largely unknown. To address this issue, we carried out 60× whole-genome sequencing of 26 metastases from four patients with pancreatic cancer. We found that identical mutations in known driver genes were present in every metastatic lesion for each patient studied. Passenger gene mutations, which do not have known or predicted functional consequences, accounted for all intratumoral heterogeneity. Even with respect to these passenger mutations, our analysis suggests that the genetic similarity among the founding cells of metastases was higher than that expected for any two cells randomly taken from a normal tissue. The uniformity of known driver gene mutations among metastases in the same patient has critical and encouraging implications for the success of future targeted therapies in advanced-stage disease.


Assuntos
Mutação/genética , Metástase Neoplásica/genética , Neoplasias Pancreáticas/genética , Humanos
12.
Science ; 357(6349): 409-413, 2017 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-28596308

RESUMO

The genomes of cancers deficient in mismatch repair contain exceptionally high numbers of somatic mutations. In a proof-of-concept study, we previously showed that colorectal cancers with mismatch repair deficiency were sensitive to immune checkpoint blockade with antibodies to programmed death receptor-1 (PD-1). We have now expanded this study to evaluate the efficacy of PD-1 blockade in patients with advanced mismatch repair-deficient cancers across 12 different tumor types. Objective radiographic responses were observed in 53% of patients, and complete responses were achieved in 21% of patients. Responses were durable, with median progression-free survival and overall survival still not reached. Functional analysis in a responding patient demonstrated rapid in vivo expansion of neoantigen-specific T cell clones that were reactive to mutant neopeptides found in the tumor. These data support the hypothesis that the large proportion of mutant neoantigens in mismatch repair-deficient cancers make them sensitive to immune checkpoint blockade, regardless of the cancers' tissue of origin.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Biomarcadores Tumorais/antagonistas & inibidores , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/terapia , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/terapia , Síndromes Neoplásicas Hereditárias/imunologia , Síndromes Neoplásicas Hereditárias/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/imunologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Reparo de Erro de Pareamento de DNA , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Síndromes Neoplásicas Hereditárias/genética , Síndromes Neoplásicas Hereditárias/mortalidade , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T/imunologia , Adulto Jovem
13.
CNS Oncol ; 4(6): 393-8, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26507609

RESUMO

Treatment of patients with newly diagnosed primary CNS lymphomas using high dose methotrexate regimens is reported to yield about 30% long term survivors with minimal neurotoxicity. As in other systemic large cell lymphomas, it is generally assumed that most relapses occur within 5 years of diagnosis. A retrospective review of the Johns Hopkins experience in 52 patients treated between 1995 and 2008 yielded 19 patients (37%) who achieved a complete response and were followed for over 5 years. Four of these patients remained progression-free for over 10 years. However, two of these long-term survivors have now relapsed over 10 years after their initial diagnosis. An analysis of progression and overall survival does not reveal a plateau suggesting that even patients who have not recurred for over 10 years remain at high risk for relapse after treatment with single agent high dose methotrexate.


Assuntos
Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Linfoma/tratamento farmacológico , Metotrexato/administração & dosagem , Adulto , Idoso , Intervalo Livre de Doença , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Resultado do Tratamento
14.
Hum Gene Ther ; 23(9): 917-31, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22734618

RESUMO

We evaluated the potential of an anti-human immunodeficiency virus (HIV) Tat intrabody (intracellular antibody) to promote the survival of CD4(+) cells after chimeric simian immunodeficiency virus (SIV)/HIV (SHIV) infection in rhesus macaques. Following optimization of stimulation and transduction conditions, purified CD4(+) T cells were transduced with GaLV-pseudotyped retroviral vectors expressing either an anti-HIV-1 Tat or a control single-chain intrabody. Ex vivo intrabody-gene marking was highly efficient, averaging four copies per CD4(+) cell. Upon reinfusion of engineered autologous CD4(+) cells into two macaques, high levels of gene marking (peak of 0.6% and 6.8% of peripheral blood mononuclear cells (PBMCs) and 0.3% or 2.2% of the lymph node cells) were detected in vivo. One week post cell infusion, animals were challenged with SHIV 89.6p and the ability of the anti-HIV Tat intrabody to promote cell survival was evaluated. The frequency of genetically modified CD4(+) T cells progressively decreased, concurrent with loss of CD4(+) cells and elevated viral loads in both animals. However, CD4(+) T cells expressing the therapeutic anti-Tat intrabody exhibited a relative survival advantage over an 8- and 21-week period compared with CD4(+) cells expressing a control intrabody. In one animal, this survival benefit of anti-Tat transduced cells was associated with a reduction in viral load. Overall, these results indicate that a retrovirus-mediated anti-Tat intrabody provided significant levels of gene marking in PBMCs and peripheral tissues and increased relative survival of transduced cells in vivo.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Gammaretrovirus , Anticorpos Anti-HIV/biossíntese , HIV-1 , Anticorpos de Cadeia Única/biossíntese , Transdução Genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Animais , Linfócitos T CD4-Positivos/imunologia , Sobrevivência Celular , Expressão Gênica , Anticorpos Anti-HIV/genética , Humanos , Transfusão de Linfócitos , Macaca mulatta , Anticorpos de Cadeia Única/genética , Fatores de Tempo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/imunologia
15.
J Immunol Methods ; 367(1-2): 95-8, 2011 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-21315723

RESUMO

Since the vast majority of infections occur at mucosal surfaces, accurate characterization of mucosal immune cells is critically important for understanding transmission and control of infectious diseases. Standard flow cytometric analysis of cells obtained from mucosal tissues can provide valuable information on the phenotype of mucosal leukocytes and their relative abundance, but does not provide absolute cell counts of mucosal cell populations. We developed a bead-based flow cytometry assay to determine the absolute numbers of multiple mononuclear cell types in colorectal biopsies of rhesus macaques. Using 10-color flow cytometry panels and pan-fluorescent beads, cells were enumerated in biopsy specimens by adding a constant ratio of beads per mg of tissue and then calculating cell numbers/mg of tissue based on cell-to-bead ratios determined at the time of sample acquisition. Testing in duplicate specimens showed the assay to be highly reproducible (Spearman R=0.9476, P<0.0001). Using this assay, we report enumeration of total CD45(+) leukocytes, CD4(+) and CD8(+) T cells, B cells, NK cells, CD14(+) monocytes, and myeloid and plasmacytoid dendritic cells in colorectal biopsies, with cell numbers in normal rhesus macaques varying from medians of 4486 cells/mg (T cells) to 3 cells/mg (plasmacytoid dendritic cells). This assay represents a significant advancement in rapid, accurate quantification of mononuclear cell populations in mucosal tissues and could be applied to provide absolute counts of a variety of different cell populations in diverse tissues.


Assuntos
Citometria de Fluxo/métodos , Leucócitos Mononucleares/citologia , Animais , Fluorescência , Antígenos HLA-DR/análise , Antígenos Comuns de Leucócito/análise , Contagem de Leucócitos , Macaca mulatta , Mucosa/citologia , Reprodutibilidade dos Testes
16.
PLoS One ; 5(8): e12357, 2010 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-20808813

RESUMO

Although a variety of genetic strategies have been developed to inhibit HIV replication, few direct comparisons of the efficacy of these inhibitors have been carried out. Moreover, most studies have not examined whether genetic inhibitors are able to induce a survival advantage that results in an expansion of genetically-modified cells following HIV infection. We evaluated the efficacy of three leading genetic strategies to inhibit HIV replication: 1) an HIV-1 tat/rev-specific small hairpin (sh) RNA; 2) an RNA antisense gene specific for the HIV-1 envelope; and 3) a viral entry inhibitor, maC46. In stably transduced cell lines selected such that >95% of cells expressed the genetic inhibitor, the RNA antisense envelope and viral entry inhibitor maC46 provided the strongest inhibition of HIV-1 replication. However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells. The selective advantage of the maC46 fusion inhibitor was also observed in HIV-1-infected cultures of primary T lymphocytes as well as in HIV-1-infected humanized mice. These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo. Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes.


Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Membrana Celular/metabolismo , Inibidores da Fusão de HIV/metabolismo , Infecções por HIV , HIV-1/fisiologia , Proteínas Recombinantes de Fusão/genética , Animais , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Sobrevivência Celular , Expressão Gênica , Terapia Genética , Inibidores da Fusão de HIV/farmacologia , Infecções por HIV/genética , Infecções por HIV/terapia , HIV-1/efeitos dos fármacos , Humanos , Camundongos , RNA Antissenso/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Especificidade da Espécie , Transdução Genética , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
17.
Mol Ther ; 12(6): 1157-67, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16168713

RESUMO

We examined the ability of a HIV-1-based vector (VRX494) encoding a 937-bp antisense HIV-1 envelope sequence to inhibit the replication of chimeric SIV/HIV-1 viruses encoding the HIV-1 envelope. Challenge of VRX494-transduced CEMx174 cells resulted in potent inhibition of HIV-1 and several SHIV strains. To evaluate the potential efficacy of the VRX494 vector for stem cell gene therapy, rhesus CD34(+) bone marrow cells were transduced with VRX494 and then cultured on thymus stroma to induce T cell differentiation. Transduction conditions for CD34(+) cells were optimized to yield high transduction efficiency with minimal effective multiplicity of infection. Purified CD4(+) GFP(+) T cells derived from VRX494-transduced CD34(+) cells strongly inhibited SHIV HXBC2P 3.2 and SHIV 89.6P replication compared to controls. Southern blot analysis of VRX494-transduced T cell clones revealed a subset of cells with multiple proviral copies per cell. Expression of GFP and the antisense inhibitor in VRX494-transduced cells was upregulated by Tat. Analysis of HIV-1 envelope sequences in VRX494-transduced cells revealed modifications consistent with those mediated by double-stranded RNA-dependent adenosine deaminase. These results indicate that the macaque/SHIV model should serve as a useful preclinical model to evaluate this lentiviral vector expressing an HIV-1 antisense inhibitor for stem cell gene therapy for AIDS.


Assuntos
Antígenos CD34/biossíntese , Linfócitos T CD4-Positivos/imunologia , HIV-1/genética , Células-Tronco Hematopoéticas/metabolismo , Lentivirus/genética , Vírus da Imunodeficiência Símia/genética , Replicação Viral , Adenosina Desaminase/metabolismo , Animais , Antígenos CD34/genética , Southern Blotting , Células da Medula Óssea/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular , Linhagem Celular , Relação Dose-Resposta a Droga , Citometria de Fluxo , Produtos do Gene env/metabolismo , Produtos do Gene rev/metabolismo , Produtos do Gene tat/metabolismo , Terapia Genética/métodos , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Hematopoéticas/virologia , Humanos , Leucócitos Mononucleares/metabolismo , Macaca mulatta , Modelos Genéticos , Oligonucleotídeos Antissenso/química , RNA/química , Retroviridae/genética , Células-Tronco/metabolismo , Linfócitos T/metabolismo , Regulação para Cima , Produtos do Gene rev do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência Humana
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