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Glycans are inherently heterogeneous, yet glycosylation is essential in eukaryotes, and glycans show characteristic cell type-dependent distributions. By using an immortalized human mesenchymal stromal cell (MSC) line model, we show that both N- and O-glycan processing in the Golgi functionally modulates early steps of osteogenic differentiation. We found that inhibiting O-glycan processing in the Golgi prior to the start of osteogenesis inhibited the mineralization capacity of the formed osteoblasts 3 weeks later. In contrast, inhibition of N-glycan processing in MSCs altered differentiation to enhance the mineralization capacity of the osteoblasts. The effect of N-glycans on MSC differentiation was mediated by the phosphoinositide-3-kinase (PI3K)/Akt pathway owing to reduced Akt phosphorylation. Interestingly, by inhibiting PI3K during the first 2 days of osteogenesis, we were able to phenocopy the effect of inhibiting N-glycan processing. Thus, glycan processing provides another layer of regulation that can modulate the functional outcome of differentiation. Glycan processing can thereby offer a novel set of targets for many therapeutically attractive processes.
Assuntos
Diferenciação Celular/genética , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética , Polissacarídeos/metabolismo , Calcificação Fisiológica/genética , Linhagem Celular , Glicosilação , Complexo de Golgi/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Osteoblastos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Vaccines against hepatitis B virus confer effective protection. Enzyme-linked immunosorbent assay was developed to test for specific antibodies in female genital tract secretions. Anti-hepatitis B IgG and IgA were detected in the cervicovaginal secretions of women after hepatitis B vaccination, indicating a potential genital tract role for neutralizing antibodies against sexually transmitted hepatitis B virus.
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Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Hepatite B/prevenção & controle , Vacinação , Adulto , Formação de Anticorpos , Feminino , Genitália Feminina/imunologia , Genitália Feminina/virologia , Hepatite B/imunologia , Hepatite B/virologia , Humanos , Hímen/imunologia , Hímen/virologia , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Adulto JovemRESUMO
BACKGROUND: In order to conduct laboratory studies on donated cervical tissue at suitable times an effective and reliable cryopreservation protocol for cervical tissue is required. METHODS: An active freezing approach was devised utilising 10% dimethyl sulfoxide in foetal bovine serum as a cryoprotective agent with a cooling rate of 1 °C/min to -50 °C then 10 °C/min to -120 °C; a related thawing protocol was also optimised which would allow for the bio-banking of cervical tissue. Viability of freshly harvested cervical tissue was compared to frozen-thawed samples utilising colorimetric MTT assay. In parallel, fresh and freeze-thawed samples were cultured and tested on days 1, 7 and 14 to determine whether bio-banking had detrimental effects on tissue viability over time. RESULTS: Repeat testing revealed that tissue viability between fresh and freeze-thawed samples was comparable at all four time points (days 0, 1, 7 and 14) with no apparent reductions of viability, thus demonstrating this method of cryopreserving cervical tissue is reliable and reproducible, without detrimental effects on live tissue culture. We believe this methodology creates the opportunity for bio-banking donated cervical tissues, which aids improved experimental design and reduces time pressures and wastage.
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Colo do Útero , Criopreservação/métodos , Preservação de Órgãos/métodos , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Feminino , Congelamento , Humanos , SoroRESUMO
BACKGROUND: Human T-lymphotropic virus type 1 (HTLV-1) infects an estimated 10 million persons globally with transmission resulting in lifelong infection. Disease, linked to high proviral load, occurs in a minority. In established infection HTLV-1 replicates through infectious spread and clonal expansion of infected lymphocytes. Little is known about acute HTLV-1 infection. The kinetics of early HTLV-1 infection, following transplantation-acquired infection in three recipients from one HTLV-1 infected donor, is reported. The recipients were treated with two HTLV-1 enzyme inhibitors 3 weeks post exposure following the detection of HTLV-1 provirus at low level in each recipient. HTLV-1 infection was serially monitored by serology, quantification of proviral load and HTLV-1 2LTR DNA circles and by HTLV-1 unique integration site analysis. RESULTS: HTLV-1 antibodies were first detected 16-39 days post-transplantation. HTLV-1 provirus was detected by PCR on day 16-23 and increased by 2-3 log by day 38-45 with a peak proviral doubling time of 1.4 days, after which steady state was reached. The rapid proviral load expansion was associated with high frequency of HTLV-1 2LTR DNA circles. The number of HTLV-1 unique integration sites was high compared with established HTLV-1 infection. Clonal expansion of infected cells was detected as early as day 37 with high initial oligoclonality index, consistent with early mitotic proliferation. CONCLUSIONS: In recipients infected through organ transplantation HTLV-1 disseminated rapidly despite early anti-HTLV-1 treatment. Proviral load set point was reached within 6 weeks. Seroconversion was not delayed. Unique integration site analysis and HTLV-1 2LTR DNA circles indicated early clonal expansion and high rate of infectious spread.
Assuntos
Infecções por HTLV-I/patologia , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , Provírus/isolamento & purificação , Transplantados , Transplante/efeitos adversos , Carga Viral , Anticorpos Antivirais/sangue , Antivirais/uso terapêutico , DNA Viral/análise , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Humanos , Reação em Cadeia da Polimerase , Fatores de TempoRESUMO
OBJECTIVES: Human T-lymphotropic virus (HTLV)-1 causes T-cell leukaemia and myelopathy. Together with HTLV-2, it is endemic in some African nations. Seroprevalence data from Malawi are scarce, with no reports on associated disease incidence. HTLV seroprevalence and type were tested in 418 healthy mothers from Malawi. In addition, we tested the sera of 534 children to investigate mother-to-child transmission. To provide context, we conducted a systematic review and meta-analysis of HTLV seroprevalence in African women and children. METHODS: Stored samples from a previous childhood cancer and BBV study were analysed. ELISA was used for HTLV screening followed by immunoblot for confirmation and typing. Standard methods were used for the systematic review. RESULTS: HTLV seroprevalence was 2.6% (11/418) in mothers and 2.2% (12/534) in children. Three mothers carried HTLV-1 alone, seven had HTLV-2 and one was dually infected. Three children carried HTLV-1 alone, seven had HTLV-2 and two were dually infected. Only two corresponding mothers of the 12 HTLV-positive children were HTLV positive. The systematic review included 66 studies of women and 13 of children conducted in 25 African countries. Seroprevalence of HTLV-1 varied from 0 to 17% and of HTLV-2 from 0 to 4%. CONCLUSIONS: In contrast to findings from other studies in Africa, the seroprevalence of HTLV-2 was higher than that of HTLV-1 in Malawi and one of the highest for the African region. The lack of mother-child concordance suggests alternative sources of infection among children. Our data and analyses contribute to HTLV prevalence mapping in Africa.
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Infecções por HTLV-I/epidemiologia , Infecções por HTLV-II/epidemiologia , Vírus Linfotrópico T Tipo 1 Humano , Vírus Linfotrópico T Tipo 2 Humano , Adolescente , África/epidemiologia , Criança , Pré-Escolar , Coinfecção/epidemiologia , Feminino , Humanos , Lactente , Transmissão Vertical de Doenças Infecciosas/estatística & dados numéricos , Malaui/epidemiologia , Masculino , Gravidez , Complicações Infecciosas na Gravidez/epidemiologia , Estudos SoroepidemiológicosRESUMO
Angiogenesis is an essential process during tissue regeneration; however, the amount of angiogenesis directly correlates with the level of wound scarring. Angiogenesis is lower in scar-free foetal wounds while angiogenesis is raised and abnormal in pathophysiological scarring such as hypertrophic scars and keloids. Delineating the mechanisms that modulate angiogenesis and could reduce scarring would be clinically useful. Beta-adrenoceptors (ß-AR) are G protein-coupled receptors (GPCRs) expressed on all skin cell-types. They play a role in wound repair but their specific role in angiogenesis is unknown. In this study, a range of in vitro assays (single cell migration, scratch wound healing, ELISAs for angiogenic growth factors and tubule formation) were performed with human dermal microvascular endothelial cells (HDMEC) to investigate and dissect mechanisms underpinning ß-AR-mediated modulation of angiogenesis in chick chorioallantoic membranes (CAM) and murine excisional skin wounds. ß-AR activation reduced HDMEC migration via cyclic adenosine monophosphate (cAMP)-dependent and protein kinase A (PKA)-independent mechanisms as demonstrated through use of an EPAC agonist that auto-inhibited the cAMP-mediated ß-AR transduced reduction in HDMEC motility; a PKA inhibitor was, conversely, ineffective. ELISA studies demonstrated that ß-AR activation reduced pro-angiogenic growth factor secretion from HDMECs (fibroblast growth factor 2) and keratinocytes (vascular endothelial growth factor A) revealing possible ß-AR-mediated autocrine and paracrine anti-angiogenic mechanisms. In more complex environments, ß-AR activation delayed HDMEC tubule formation and decreased angiogenesis both in the CAM assay and in murine excisional skin wounds in vivo. ß-AR activation reduced HDMEC function in vitro and angiogenesis in vivo; therefore, ß-AR agonists could be promising anti-angiogenic modulators in skin.
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AMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Neovascularização Patológica/metabolismo , Receptores Adrenérgicos beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/fisiologia , Animais , Movimento Celular , Células Cultivadas , Células Endoteliais/citologia , Humanos , Camundongos , Pele/citologia , Pele/metabolismoRESUMO
OBJECTIVES: Hepatitis C virus (HCV) prevalence is poorly mapped in the East African region; with the advent of novel HCV therapies, better epidemiological data are required to target the infection. We sought to estimate HCV prevalence in healthy Malawian mothers and assess mother-to-child transmission (MTCT); context is provided by reviewing previously published HCV prevalence data from the region. METHODS: Using ELISA screening and confirmatory blot, serological testing of 418 healthy Malawian mothers for HCV was performed. To examine MTCT, the children of any positive women were also tested for HCV; all children had malignant disease unrelated to hepatocellular carcinoma. We compared our results to published literature on HCV prevalence in Malawi and its neighbouring countries. RESULTS: Three of 418 women were HCV reactive by ELISA; two were confirmed positive by immunoblot (0.5%). One child of an HCV-infected mother was HCV seropositive. The literature review revealed HCV prevalence ranging from 0 to 7.2% in the region, being highest in Tanzania and specifically for cohorts of inpatients and HIV-co-infected people. The overall estimated prevalence of HCV in Malawi was 1.0% (95%CI 0.7-1.4) when all studies were included (including this one), but lower in healthy cohorts alone at 0.3% (95%CI 0.1-1.2). CONCLUSIONS: This is the first study using confirmatory tests to examine HCV prevalence in healthy Malawian mothers; the prevalence was low. Future studies need to address the source of infection in healthy women.
RESUMO
During innate immune responses, the inflammatory CC chemokine receptors CCR1, CCR2, and CCR5 mediate the recruitment of blood monocytes to infected tissues by promoting cell migration in response to chemokines CCL2-5. Toll-like receptors also play an essential role, allowing pathogen recognition by the recruited monocytes. Here, we demonstrate that Toll-like receptor 2 (TLR2) stimulation by lipoteichoic acid (LTA) from Staphylococcus aureus leads to gradual down-modulation of CCR1, CCR2, and CCR5 from the plasma membrane of human blood-isolated monocytes and inhibits chemotaxis. Interestingly, LTA does not promote rapid desensitization of chemokine-mediated calcium responses. We found that the TLR2 crosstalk with chemokine receptors is not dependent on the Toll/interleukin-1 receptor domain-containing adaptor protein, but instead involves phospholipase C, the small G protein Rac1, and is phorbol ester sensitive. Activation of this pathway by LTA lead to ß-arrestin-mediated endocytosis of Ser349-phosphorylated CCR5 into recycling endosomes, as does CCL5 treatment. However, LTA-induced internalization of CCR5 is a slower process associated with phospholipase C-mediated and phorbol ester-sensitive phosphorylation. Overall, our data indicate that TLR2 negatively regulates CCR1, CCR2, and CCR5 on human blood monocytes by activating the machinery used to support chemokine-dependent down-modulation and provide a molecular mechanism for inhibiting monocyte migration after pathogen recognition.
Assuntos
Monócitos/imunologia , Receptores CCR1/sangue , Receptores CCR2/sangue , Receptores CCR5/sangue , Receptor 2 Toll-Like/sangue , Sinalização do Cálcio/efeitos dos fármacos , Movimento Celular/imunologia , Quimiotaxia de Leucócito , Regulação para Baixo/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Técnicas In Vitro , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , Fosforilação , Receptor Cross-Talk/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Staphylococcus aureus/imunologia , Ácidos Teicoicos/imunologia , Ácidos Teicoicos/farmacologiaRESUMO
OBJECTIVE: To compare efficiency and cost-effectiveness of an observation unit (OU) when managed as a closed unit vs an open unit. METHODS: This observational, retrospective study of a 30-bed OU compared three time periods: Nov 2007 to Aug 2008 (period 1), Nov 2008 to Aug 2009 (period 2) and Nov 2010 to Aug 2011 (period 3). The OU was managed and staffed by non-emergency department physicians as an open unit during period 1, and a closed unit by emergency department physicians during periods 2 and 3. RESULTS: OU volume was greatest in period 3 (1 vs 3, 95% CI -235.8 to -127.9; 2 vs 3, 95% CI -191.9 to -84.095%). Periods 2 and 3 had shorter lengths of stay for discharged (1 vs 2, 95% CI -6.6 to 1.7; 1 vs 3, 95% CI -8.1 to -3.1) and admitted (1 vs 2, 95% CI -11.4 to -8.6; 1 vs 3, 95% CI -11.8 to -9.0) patients, less admission rates (P < .001), and less 30-day all cause admission rates after discharge (P < .0001). Cost was less during periods 2 and 3 for direct (1 vs 2, 95% CI -392.5 to -305.9; 1 vs 3, 95% CI -471.4 to -388.4), indirect (1 vs 2, 95% CI -249.5 to - 199.8; 1 vs 3, 95% CI -187 to-139.4) and total cost (1 vs 2, 95% CI -640.7 to -507; 1 vs 3, 95% CI -657.2 to -529). CONCLUSION: The same OU was more efficient and cost-effective when managed as a closed unit vs an open unit.
Assuntos
Eficiência Organizacional/estatística & dados numéricos , Serviço Hospitalar de Emergência/organização & administração , Custos Hospitalares/estatística & dados numéricos , Unidades Hospitalares/organização & administração , Hospitais de Ensino/organização & administração , Análise Custo-Benefício , Eficiência Organizacional/economia , Unidades Hospitalares/economia , Unidades Hospitalares/estatística & dados numéricos , Hospitais de Ensino/economia , Hospitais de Ensino/estatística & dados numéricos , Humanos , Tempo de Internação/economia , Tempo de Internação/estatística & dados numéricos , Michigan , Admissão do Paciente/economia , Admissão do Paciente/estatística & dados numéricos , Estudos RetrospectivosRESUMO
Gene therapy (GT) provides a potentially curative treatment option for patients with sickle cell disease (SCD); however, the occurrence of myeloid malignancies in GT clinical trials has prompted concern, with several postulated mechanisms. Here, we used whole-genome sequencing to track hematopoietic stem cells (HSCs) from six patients with SCD at pre- and post-GT time points to map the somatic mutation and clonal landscape of gene-modified and unmodified HSCs. Pre-GT, phylogenetic trees were highly polyclonal and mutation burdens per cell were elevated in some, but not all, patients. Post-GT, no clonal expansions were identified among gene-modified or unmodified cells; however, an increased frequency of potential driver mutations associated with myeloid neoplasms or clonal hematopoiesis (DNMT3A- and EZH2-mutated clones in particular) was observed in both genetically modified and unmodified cells, suggesting positive selection of mutant clones during GT. This work sheds light on HSC clonal dynamics and the mutational landscape after GT in SCD, highlighting the enhanced fitness of some HSCs harboring pre-existing driver mutations. Future studies should define the long-term fate of mutant clones, including any contribution to expansions associated with myeloid neoplasms.
Assuntos
Anemia Falciforme , Neoplasias , Humanos , Hematopoese/genética , Filogenia , Mutação/genética , Células-Tronco Hematopoéticas/patologia , Células Clonais , Anemia Falciforme/genética , Anemia Falciforme/terapia , Anemia Falciforme/patologia , Terapia Genética , Neoplasias/patologiaRESUMO
[This corrects the article DOI: 10.3389/fimmu.2022.903796.].
RESUMO
Heterogeneity of bone marrow mesenchymal stromal cells (MSCs, frequently referred to as "mesenchymal stem cells") clouds biological understanding and hampers their clinical development. In MSC cultures most commonly used in research and therapy, we have identified an MSC subtype characterized by CD317 expression (CD317pos (29.77 ± 3.00% of the total MSC population), comprising CD317dim (28.10 ± 4.60%) and CD317bright (1.67 ± 0.58%) MSCs) and a constitutive interferon signature linked to human disease. We demonstrate that CD317pos MSCs induced cutaneous tissue damage when applied a skin explant model of inflammation, whereas CD317neg MSCs had no effect. Only CD317neg MSCs were able to suppress proliferative cycles of activated human T cells in vitro, whilst CD317pos MSCs increased polarization towards pro-inflammatory Th1 cells and CD317neg cell lines did not. Using an in vivo peritonitis model, we found that CD317neg and CD317pos MSCs suppressed leukocyte recruitment but only CD317neg MSCs suppressed macrophage numbers. Using MSC-loaded scaffolds implanted subcutaneously in immunocompromised mice we were able to observe tissue generation and blood vessel formation with CD317neg MSC lines, but not CD317pos MSC lines. Our evidence is consistent with the identification of an immune stromal cell, which is likely to contribute to specific physiological and pathological functions and influence clinical outcome of therapeutic MSCs.
Assuntos
Células-Tronco Mesenquimais , Animais , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Transdução de Sinais , Células Estromais , Células Th1RESUMO
Co-ordinated movement and controlled positioning of leucocytes is key to the development, maintenance and proper functioning of the immune system. Chemokines and their receptors play an essential role in these events by mediating directed cell migration, often referred to as chemotaxis. The chemotactic property of these molecules is also thought to contribute to an array of pathologies where inappropriate recruitment of specific chemokine receptor-expressing leucocytes is observed, including cancer and inflammatory diseases. As a result, chemokine receptors have become major targets for therapeutic intervention, and during the past 15 years much research has been devoted to understanding the regulation of their biological activity. From these studies, processes which govern the availability of functional chemokine receptors at the cell surface have emerged as playing a central role. In this review, we summarize and discuss current knowledge on the molecular mechanisms contributing to the regulation of chemokine receptor surface expression, from gene transcription and protein degradation to post-translational modifications, multimerization, intracellular transport and cross-talk.
Assuntos
Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Regulação da Expressão Gênica , Humanos , Modelos Biológicos , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteólise , Receptor Cross-Talk , Receptores de Quimiocinas/químicaRESUMO
The chemokine CXCL4/platelet factor-4 is released by activated platelets in micromolar concentrations and is a chemoattractant for leukocytes via an unidentified receptor. Recently, a variant of the human chemokine receptor CXCR3 (CXCR3-B) was described, which transduced apoptotic but not chemotactic signals in microvascular endothelial cells following exposure to high concentrations of CXCL4. Here, we show that CXCL4 can induce intracellular calcium release and the migration of activated human T lymphocytes. CXCL4-induced chemotaxis of T lymphocytes was inhibited by a CXCR3 antagonist and pretreatment of cells with pertussis toxin (PTX), suggestive of CXCR3-mediated G-protein signaling via Galphai-sensitive subunits. Specific binding by T lymphocytes of the CXCR3 ligand CXCL10 was not effectively competed by CXCL4, suggesting that the two are allotopic ligands. We subsequently used expression systems to dissect the potential roles of each CXCR3 isoform in mediating CXCL4 function. Transient expression of the CXCR3-A and CXCR3-B isoforms in the murine pre-B cell L1.2 produced cells that migrated in response to CXCL4 in a manner sensitive to PTX and a CXCR3 antagonist. Binding of radiolabeled CXCL4 to L1.2 CXCR3 transfectants was of low affinity and appeared to be mediated chiefly by glycosaminoglycans (GAGs), as no specific CXCL4 binding was observed in GAG-deficient 745-Chinese hamster ovary cells stably expressing CXCR3. We suggest that following platelet activation, the CXCR3/CXCL4 axis may play a role in T lymphocyte recruitment and the subsequent amplification of inflammation observed in diseases such as atherosclerosis. In such a setting, antagonism of the CXCR3/CXCL4 axis may represent a useful, therapeutic intervention.
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Quimiotaxia de Leucócito/efeitos dos fármacos , Fator Plaquetário 4/farmacologia , Receptores CXCR3/fisiologia , Linfócitos T/fisiologia , Movimento Celular/efeitos dos fármacos , Separação Celular/métodos , Clonagem Molecular , Humanos , Ativação Linfocitária , Ensaio Radioligante , Receptores CXCR3/genética , Valores de Referência , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
Using the Zimbardo Time Perspective Inventory (ZTPI; P. G. Zimbardo & J. N. Boyd, 1999), the authors found that homeless people, in comparison with a control group, had a significantly more negative outlook concerning their past and present as evinced by high Past-Negative and Present-Fatalistic scores and low Past-Positive scores on the ZTPI. However, the homeless individuals were almost indistinguishable from control participants on measures of Present-Hedonism and Future thinking. The homeless individuals had significantly higher levels of depression, with 31 out of 50 (62%) reaching criteria for probable depression. However, this finding was unrelated to their atypical time perspective. There was no significant relation between substance misuse and time perspective. Despite their current difficulties, including depression and drug abuse, the homeless individuals maintained a propensity toward future thinking characterized by striving to achieve their goals.
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Transtorno Depressivo/epidemiologia , Pessoas Mal Alojadas/psicologia , Transtornos Relacionados ao Uso de Substâncias/epidemiologia , Logro , Adaptação Psicológica , Grupos Controle , Transtorno Depressivo/psicologia , Previsões , Objetivos , Pessoas Mal Alojadas/estatística & dados numéricos , Motivação , Inventário de Personalidade/estatística & dados numéricos , Transtornos Relacionados ao Uso de Substâncias/psicologia , Pensamento , Fatores de TempoRESUMO
Activated platelets release micromolar concentrations of the chemokine CXCL4/Platelet Factor-4. Deposition of CXCL4 onto the vascular endothelium is involved in atherosclerosis, facilitating monocyte arrest and recruitment by an as yet, unidentified receptor. Here, we demonstrate that CXCL4 drives chemotaxis of the monocytic cell line THP-1. Migration and intracellular calcium responses induced by CXCL4 were pertussis toxin-sensitive, implicating a GPCR in signal transduction. Cell treatment with chondroitinase ABC ablated migration, suggesting that cis presentation of CXCL4 by cell surface glycosaminoglycans to a GPCR is required. Although CXCR3 has been previously described as a CXCL4 receptor, THP-1 cells were unresponsive to CXCR3 ligands and CXCL4-induced migration was insensitive to a CXCR3 antagonist, suggesting that an alternative receptor is involved. Interrogating CC-class chemokine receptor transfectants, we unexpectedly found that CXCL4 could induce the migration of CCR1-expressing cells and also induce CCR1 endocytosis. Extending our findings to primary human monocytes, we observed that CXCL4 induced CCR1 endocytosis and could induce monocyte chemotaxis in a CCR1 antagonist-sensitive manner. Collectively, our data identify CCR1 as a previously elusive monocyte CXCL4 receptor and suggest that CCR1 may play a role in inflammation where the release of CXCL4 is implicated.
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Quimiotaxia , Monócitos/metabolismo , Fator Plaquetário 4/metabolismo , Receptores CCR1/metabolismo , Cálcio/metabolismo , Linhagem Celular , Células Cultivadas , Condroitina ABC Liase/farmacologia , Endocitose , Humanos , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , Toxina Pertussis/farmacologia , Fator Plaquetário 4/genética , Ligação ProteicaRESUMO
Clonal expansion of human T-lymphotropic virus type-1 (HTLV-1) infected cells in vivo is well documented. Unlike human immunodeficiency virus type 1 (HIV-1), HTLV-1 plasma RNA is sparse. The contribution of the "mitotic" spread of HTLV-1 compared with infectious spread of the virus to HTLV-1 viral burden in established infection is uncertain. Since extrachromosomal long terminal repeat (LTR) DNA circles are indicators of viral replication in HIV-1 carriers with undetectable plasma HIV RNA, we hypothesised that HTLV-1 LTR circles could indicate reverse transcriptase (RT) usage and infectious activity. 1LTR and 2LTR DNA circles were measured in HTLV-1 cell lines and peripheral blood mononuclear cells (PBMC) of asymptomatic carriers (ACs) and patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T cell leukaemia/lymphoma (ATLL). 1LTR DNA circles were detected in 14/20 patients at a mean of 1.38/100 PBMC but did not differentiate disease status nor correlate with HTLV-1 DNA copies. 2LTR DNA circles were detected in 30/31 patients and at higher concentrations in patients with HTLV-1-associated diseases, independent of HTLV-1 DNA load. In an incident case the 2LTR DNA circle concentration increased 2.1 fold at the onset of HAM/TSP compared to baseline. Detectable and fluctuating levels of HTLV-1 DNA circles in patients indicate viral RT usage and virus replication. Our results indicate HTLV-1 viral replication capacity is maintained in chronic infection and may be associated with disease onset.
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Biomarcadores/análise , DNA Circular/análise , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Sequências Repetidas Terminais , Replicação Viral , Sangue/virologia , Portador Sadio/virologia , Células Cultivadas , DNA Circular/genética , Feminino , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Leucócitos Mononucleares/virologia , Masculino , DNA Polimerase Dirigida por RNA/metabolismoRESUMO
CCR5 is a chemokine receptor expressed on leukocytes and a coreceptor used by HIV-1 to enter CD4(+) T lymphocytes and macrophages. Stimulation of CCR5 by chemokines triggers internalization of chemokine-bound CCR5 molecules in a process called down-modulation, which contributes to the anti-HIV activity of chemokines. Recent studies have shown that CCR5 conformational heterogeneity influences chemokine-CCR5 interactions and HIV-1 entry in transfected cells or activated CD4(+) T lymphocytes. However, the effect of CCR5 conformations on other cell types and on the process of down-modulation remains unclear. We used mAbs, some already shown to detect distinct CCR5 conformations, to compare the behavior of CCR5 on in vitro generated human T cell blasts, monocytes and MDMs and CHO-CCR5 transfectants. All human cells express distinct antigenic forms of CCR5 not detected on CHO-CCR5 cells. The recognizable populations of CCR5 receptors exhibit different patterns of down-modulation on T lymphocytes compared with myeloid cells. On T cell blasts, CCR5 is recognized by all antibodies and undergoes rapid chemokine-mediated internalization, whereas on monocytes and MDMs, a pool of CCR5 molecules is recognized by a subset of antibodies and is not removed from the cell surface. We demonstrate that this cell surface-retained form of CCR5 responds to prolonged treatment with more-potent chemokine analogs and acts as an HIV-1 coreceptor. Our findings indicate that the regulation of CCR5 is highly specific to cell type and provide a potential explanation for the observation that native chemokines are less-effective HIV-entry inhibitors on macrophages compared with T lymphocytes.
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Regulação para Baixo , Células Mieloides/metabolismo , Receptores CCR5/fisiologia , Linfócitos T/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Antígenos/sangue , Células CHO , Cricetinae , Cricetulus , Humanos , Ligantes , Microscopia de Fluorescência , Conformação Proteica , Receptores CCR5/imunologia , Receptores CCR5/metabolismoRESUMO
The aim of this study was to identify changes in bovine macrophage gene expression in response to treatment with Escherichia coli 0157:H7 lipopolysaccharide (LPS), utilizing a human gene microarray. Bovine cDNA from control and LPS-treated primary macrophages hybridized to greater than 5644 (79.8%) of the non-control gene targets on a commercially available microarray containing greater than 7075 targets (Incyte Genomics, St. Louis, MO). Of these target sequences, 44 were differentially expressed upon exposure to LPS, including 18 genes not previously reported to exist in cattle. These included a pentaxin-related gene, CASP8, TNF-induced genes, interferon-induced genes, and inhibitors of apoptosis. Using the human microarray, cDNA from bovine LPS-treated and control macrophages consistently hybridized to targets known to be expressed constitutively by macrophages, as expected given the predicted cDNA sequence homology. That this human system was accurately estimating levels of bovine transcripts was further verified by real-time quantitative reverse transcriptase polymerase chain reaction (RTQ-PCR) using bovine-specific primers. This first report of bovine-human cross-species expression profiling by microarray hybridization demonstrates the utility of this technique in bovine gene expression and discovery.