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1.
AIDS ; 33(3): 411-423, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30703069

RESUMO

BACKGROUND: Early steps of HIV infection are mediated by the binding of the envelope to mucosal receptors as α4ß7 and the C-type lectins DC-SIGN and langerin. Previously Env-specific B-cell responses have been reported in highly exposed seronegative individuals (HESN). METHOD: Here, we studied gp120-specific antibodies ability to block HIV interaction with α4ß7, DC-SIGN and/or langerinin HESN. New cell-based assays were developed to analyze whether antibodies that can alter gp120 binding to α4ß7, DC-SIGN and/or langerin are induced in HESN. A mucosal blocking score (MBS) was defined based on the ability of antibodies to interfere with gp120/α4ß7, gp120/DC-SIGN, and gp120/langerin binding. A new MBS was evaluated in a cohort of 86 HESN individuals and compared with HIV patients or HIV unexposed healthy individuals. RESULTS: Antibodies reducing gp120 binding to both α4ß7 and DC-SIGN were present in HESN serum but also in mucosal secretions, whereas antibodies from HIV patients facilitated gp120 binding to DC-SIGN. Any correlation was observed between MBS and the capacity of antibodies to neutralize infection of α4ß7 CD4 T cells with primary isolates. CONCLUSIONS: MBS is significantly associated with protection in HESN and might reflect altered HIV spreading to mucosal-associated lymphoid tissues.

2.
J Infect Dis ; 218(3): 490-503, 2018 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-29648611

RESUMO

Background: Human cytomegalovirus (HCMV) is a leading cause of virally induced congenital disorders and morbidities in immunocompromised individuals, ie, transplant, cancer, or acquired immune deficiency syndrome patients. Human cytomegalovirus infects virtually all cell types through the envelope glycoprotein complex gH/gL/gO with or without a contribution of the pentameric gH/gL/pUL128L. Together with gH/gL, the HCMV envelope glycoprotein B (gB) contributes to the viral fusion machinery. Methods: We previously showed that gB is a ligand for the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) contributing to HCMV attachment to and infection of DC-SIGN-expressing cells. However, the features of the DC-SIGN/gB interaction remain unclear. To address this point, the role of glycans on gB and the consequences of mutagenesis and antibody-mediated blockades on both partners were examined in this study. Results: We identified DC-SIGN amino acid residues involved in this interaction through an extensive mutagenesis study. We also showed the importance of high-mannose N-glycans decorating the asparagine residue at position 208, demonstrating that the antigenic domain 5 on gB is involved in the interaction with DC-SIGN. Finally, antibody-mediated blockades allowed us to identify DC-SIGN as a major HCMV attachment receptor on monocyte-derived dendritic cells. Conclusions: Taken together, these results have permitted us to fine-map the interaction between DC-SIGN and HCMV gB.

3.
J Acquir Immune Defic Syndr ; 75(1): 118-127, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28177967

RESUMO

The homing of lymphocytes to the mucosa is mainly controlled by α4ß7 integrin, and it is amplified during gut chronic inflammation, as occurs with HIV and/or inflammatory bowel diseases. We designed and applied an improved immunization strategy based on an innovative selection process to isolate new α4ß7 lymphocyte-specific monoclonal antibodies that are able to prevent their migration into inflamed gut tissues and/or to counteract HIV infection in vitro. First, 5 monoclonal antibodies (1 IgA, 1 IgM, and 4 IgGs) were selected based on their capacity to recognize α4 or ß7 homodimers and α4ß7 heterodimers in transfected human cells. Their ability to block gp120/α4ß7 or MAdCAM-1/α4ß7 interactions was then measured in vitro with human T and B lymphocytes. In vitro, the anti-α4ß7 IgA isotype was found to have the highest affinity for the α4ß7 heterodimer, and it significantly reduced HIV replication in retinoic acid-treated α4ß7 CD4 human T cells. This α4ß7-specific IgA also displayed a high avidity for human and mouse α4ß7 lymphocytes in both mouse and human inflammatory colitis tissues. These new antibodies, and in particular those with mucosa-targeting isotypes such as IgA, could therefore be potential novel therapeutic tools for treating HIV and inflammatory bowel disease.


Assuntos
Linfócitos B/imunologia , Isotipos de Imunoglobulinas/imunologia , Integrinas/antagonistas & inibidores , Integrinas/imunologia , Linfócitos T/imunologia , Linfócitos T/virologia , Replicação Viral , Animais , Fármacos Anti-HIV/farmacologia , Anti-Inflamatórios/farmacologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , HIV/fisiologia , Humanos , Fatores Imunológicos/farmacologia , Camundongos Endogâmicos BALB C
5.
Immunobiology ; 221(1): 12-22, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26345430

RESUMO

Dogs with lymphoma are established as good model for human non-Hodgkin lymphoma studies. Canine cell lines derived from lymphomas may be valuable tools for testing new therapeutic drugs. In this context, we established a canine T-cell line, PER-VAS, from a primary aggressive T-cell lymphoma with large granular morphology. Flow cytometric analysis revealed a stable immunophenotype: PER-VAS cells were positively labelled for CD5, CD45, MHC II and TLR3, and were negative for CD3, CD4 and CD8 expression. Although unstable along the culture process, IL-17 and MMP12 proteins were detectable as late as at passages 280 and 325i.e. respectively 24 and 29 months post isolation. At passage 325, PER-VAS cells maintained the expression of IL-17, CD3, CD56, IFNγ and TNFα mRNAs as shown by RT-PCR analysis. Stable rearrangement of the TCRγ gene has been evidenced by PCR. PER-VAS cells have a high proliferation index with a doubling time of 16.5h and were tumorigenic in Nude mice. Compared to the canine cell lines already reported, PER-VAS cells display an original expression pattern, close to NKT cells, which makes them valuable tools for in vitro comparative research on lymphomas.


Assuntos
Linhagem Celular/imunologia , Expressão Gênica/imunologia , Linfoma de Células T/imunologia , RNA Mensageiro/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Linhagem Celular/patologia , Cães , Efeito Fundador , Rearranjo Gênico da Cadeia gama dos Receptores de Antígenos dos Linfócitos T , Humanos , Imunofenotipagem , Interferon gama/genética , Interferon gama/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Linfoma de Células T/genética , Linfoma de Células T/patologia , Masculino , Camundongos , Camundongos Nus , RNA Mensageiro/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/patologia , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
6.
J Immunol ; 190(2): 764-73, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-23255358

RESUMO

TLR3 belongs to the family of intracellular TLRs that recognize nucleic acids. Endolysosomal localization and cleavage of intracellular TLRs play pivotal roles in signaling and represent fail-safe mechanisms to prevent self-nucleic acid recognition. Indeed, cleavage by cathepsins is required for native TLR3 to signal in response to dsRNA. Using novel Abs generated against TLR3, we show that the conserved loop exposed in LRR12 is the single cleavage site that lies between the two dsRNA binding sites required for TLR3 dimerization and signaling. Accordingly, we found that the cleavage does not dissociate the C- and N-terminal fragments, but it generates a very stable "cleaved/associated" TLR3 present in endolysosomes that recognizes dsRNA and signals. Moreover, comparison of wild-type, noncleavable, and C-terminal-only mutants of TLR3 demonstrates that efficient signaling requires cleavage of the LRR12 loop but not dissociation of the fragments. Thus, the proteolytic cleavage of TLR3 appears to fulfill function(s) other than separating the two fragments to generate a functional receptor.


Assuntos
Transdução de Sinais , Receptor 3 Toll-Like/metabolismo , Sítios de Ligação , Catepsinas/metabolismo , Linhagem Celular , Perfilação da Expressão Gênica , Complexo de Golgi/metabolismo , Humanos , Lisossomos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteólise , Receptor 3 Toll-Like/genética
7.
BMC Cancer ; 11: 213, 2011 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-21624121

RESUMO

BACKGROUND: Chemokines and chemokine receptors are major actors of leukocytes trafficking and some have been shown to play an important role in cancer metastasis. Chemokines CCL19, CCL20 and CCL21 and their receptors CCR6 and CCR7, were assessed as potential biomarkers of metastatic dissemination in primary breast cancer. METHODS: Biomarker expression levels were evaluated using immunohistochemistry on paraffin-embedded tissue sections of breast cancer (n = 207). RESULTS: CCR6 was expressed by tumor cells in 35% of cases. CCR7 was expressed by spindle shaped stromal cells in 43% of cases but not by tumor cells in this series. CCL19 was the only chemokine found expressed in a significant number of breast cancers and was expressed by both tumor cells and dendritic cells (DC). CCR6, CCL19 and CCR7 expression correlated with histologic features of aggressive disease. CCR6 expression was associated with shorter relapse-free survival (RFS) in univariate and but not in multivariate analysis (p = 0.0316 and 0.055 respectively), and was not associated with shorter overall survival (OS). Expression of CCR7 was not significantly associated with shorter RFS or OS. The presence of CCL19-expressing DC was associated with shorter RFS in univariate and multivariate analysis (p = 0.042 and 0.020 respectively) but not with shorter OS. CONCLUSION: These results suggest a contribution of CCR6 expression on tumor cells and CCL19-expressing DC in breast cancer dissemination. In our series, unlike what was previously published, CCR7 was exclusively expressed on stromal cells and was not associated with survival.


Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/fisiopatologia , Regulação Neoplásica da Expressão Gênica , Ligantes , Receptores CCR6/metabolismo , Receptores CCR7/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/mortalidade , Quimiocinas C/metabolismo , Feminino , Humanos , Linfonodos/metabolismo , Linfonodos/patologia , Metástase Linfática/patologia , Pessoa de Meia-Idade , Prognóstico , Células Estromais/metabolismo , Células Estromais/patologia , Análise de Sobrevida
8.
Vaccine ; 29(20): 3655-61, 2011 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-21439318

RESUMO

Peyer's patch have been extensively studied as a major inductive site for mucosal immunity within the small intestine. The intestinal mucosa contains numerous dendritic cells, which induce either protective immunity to infectious agents or tolerance to innocuous antigens, including food and commensal bacteria. Although during the past few years, several subsets of human mucosal dendritic cells have been described, a precise characterization of the different mouse mucosal dendritic cells subpopulations remains to be achieved with regard to their phenotype and localization in Peyer's patch. In this report, we have investigated by immunofluorescence on cryosection and by flow cytometry, the phenotype and the localization of dendritic cells into Peyer's patch of C57Bl/6 mouse intestine using dendritic cells markers. Positive and double staining for CD11c and BDCA-2, pDC/IPC, DC-LAMP, DC-SIGN, TLR8 and Langerin have been observed revealing new mouse intestinal DC subsets. This study provides new insight in the understanding of mucosal immune responses induced by natural processes as infections but also new perspectives for the evaluation of oral vaccines.


Assuntos
Células Dendríticas/citologia , Imunidade nas Mucosas , Mucosa Intestinal/citologia , Nódulos Linfáticos Agregados/citologia , Linfócitos T/imunologia , Animais , Antígenos de Superfície/imunologia , Antígeno CD11c/imunologia , Moléculas de Adesão Celular/imunologia , Diferenciação Celular , Células Dendríticas/classificação , Células Dendríticas/imunologia , Mucosa Intestinal/imunologia , Lectinas Tipo C/imunologia , Lectinas de Ligação a Manose/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Superfície Celular/imunologia , Receptor 8 Toll-Like/imunologia
9.
Immunol Lett ; 135(1-2): 165-72, 2011 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-21078343

RESUMO

DC-SIGN is a C-type lectin of recognized importance in immunology and in the pathogenicity human pathogens. Monoclonal antibodies directed against DC-SIGN have been generated, but their systemic characterization for interfering with binding of the HIV-1 glycoprotein 120 has often been omitted. Moreover, so far, no anti-DC-SIGN monoclonal antibody has been described that recognizes its antigen after formalin fixation and paraffin embedding. In this study, we have generated new anti-DC-SIGN monoclonal antibodies using HeLa cells stably expressing DC-SIGN as immunogen. We have obtained 11 hybridoma clones producing antibodies that recognized DC-SIGN on monocyte-derived dendritic cells and on dermal-type macrophages. Seven monoclonal antibodies displayed a capacity to interfere with DC-SIGN binding to HIV-1 gp120. One recognized DC-SIGN on formalin-fixed dendritic cells and macrophages. Using this antibody we have obtained specific labelling of DC-SIGN and colocalisation with the dermal macrophage marker CD163 on human skin. The described monoclonal anti-human DC-SIGN antibodies will be of use to the scientific community to address fundamental immunology issues, in particular concerning macrophages and dendritic cells, and help elucidate infection events of pathogen targeting DC-SIGN as recognition receptor.


Assuntos
Anticorpos Bloqueadores/imunologia , Anticorpos Monoclonais Murinos/imunologia , Moléculas de Adesão Celular/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Lectinas Tipo C/imunologia , Receptores de Superfície Celular/imunologia , Animais , Anticorpos Bloqueadores/genética , Anticorpos Monoclonais Murinos/genética , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Moléculas de Adesão Celular/genética , Células Dendríticas/imunologia , Derme/imunologia , Fixadores/química , Formaldeído/química , Proteína gp120 do Envelope de HIV/genética , Células HeLa , Humanos , Lectinas Tipo C/genética , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/imunologia , Células NIH 3T3 , Receptores de Superfície Celular/genética
10.
J Neurosci Methods ; 192(2): 268-76, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20709102

RESUMO

Analyses using antibodies directed against α-synuclein play a key role in the understanding of the pathologies associated with neurodegenerative disorders such as Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). However, the generation of antibodies against immunogens with significant sequence similarity to host proteins such as α-synuclein is often hindered by host immunotolerance. In contrast to wild-type C57BL/6J and BALB/c mice immunized with recombinant human α-synuclein, C57BL/6S Δsnca mice presenting a natural deletion of the α-synuclein locus, bypassed the immunotolerance process which resulted in a much higher polyclonal antibody response. The native or fibrillized conformation of α-synuclein used as the immunogen did not have an impact on the amounts of specific antibodies in sera of the host. The immunization protocols resulted in the generation of the IgG AS11, raised against fibrillized recombinant human α-synuclein in C57BL/6S Δsnca mice. This monoclonal antibody, recognizing an N-terminal α-synuclein epitope, was selected for its specificity and significant reactivity in Western-blot, immunofluorescence and immunohistochemistry assays. The ability of AS11 to detect both soluble and aggregated forms of α-synuclein present in pathological cytoplasmic inclusions was further assessed using analysis of human brains with PD or MSA, transgenic mouse lines expressing A53T human α-synuclein, and cellular models expressing human α-synuclein. Taken together, our study indicates that novel antibodies helpful to characterize alterations of α-synuclein leading to neurodegeneration in PD and related disorders could be efficiently developed using this original immunization strategy.


Assuntos
Anticorpos Monoclonais/biossíntese , alfa-Sinucleína/imunologia , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Linhagem Celular , Células Cultivadas , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , alfa-Sinucleína/genética
11.
Vet Immunol Immunopathol ; 118(1-2): 134-9, 2007 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-17521746

RESUMO

Toll-like receptors (TLRs) are a family of functionally important receptors for recognition of pathogen-associated molecular pattern (PAMP) since they trigger the pro-inflammatory response and upregulation of costimulatory molecules, linking the rapid innate response to adaptative immunity. In human leukocytes, TLR3 has been found to be specifically expressed in dendritic cells (DC). This study examined the expression of TLR3 in canine monocytes-derived DC (cMo-DC) and PBMC using three new anti-TLR3 mAbs (619F7, 722E2 and 713E4 clones). The non-adherent cMo-DC generated after culture in canine IL-4 plus canine GM-CSF were labelled with the three anti-TLR3 clones by flow cytometry, with a strong expression shown for 619F7 and 722E2 clones. By contrast, TLR3 expression was low to moderate in canine monocytes and lymphocytes. These results were confirmed by Western blot using 619F7 and 722E2 clones and several polypeptide bands were observed, suggesting a possible cleavage of TLR3 molecule or different glycosylation states. In addition, TLR3 was detectable in immunocytochemistry by using 722E2 clone. In conclusion, this first approach to study canine TLR3 protein expression shows that three anti-TLR3 clones detect canine TLR3 and can be used to better characterize canine DC and the immune system of dogs.


Assuntos
Células Dendríticas/citologia , Células Dendríticas/metabolismo , Monócitos/citologia , Receptor 3 Toll-Like/metabolismo , Animais , Anticorpos Monoclonais , Biomarcadores/metabolismo , Células Cultivadas , Cães , Feminino , Humanos , Imuno-Histoquímica , Linfócitos , Masculino , Receptor 3 Toll-Like/genética
12.
Am J Pathol ; 168(2): 453-65, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16436660

RESUMO

Originally implicated in axon guidance, semaphorins represent a large family of molecules that are now known to be expressed in the immune system. Among different semaphorins tested by reverse transcriptase-polymerase chain reaction in human immune cells, the expression of class 6 transmembrane semaphorin SEMA6A was restricted to dendritic cells (DCs). Using in-house generated monoclonal antibodies, SEMA6A expression appeared further restricted to Langerhans cells (LCs). In vivo, SEMA6A mRNA was expressed in freshly isolated skin LCs but SEMA6A protein was not detectable on normal skin and tonsillar epithelium. Of interest, SEMA6A protein was strongly expressed on skin and bone LCs and on LCs in draining lymph nodes from patients with LC histiocytosis or dermatopathic lymphadenitis, respectively, representing two inflammatory conditions in which LCs display an immature DC-LAMP(low), CD83(low), and CCR7+ phenotype. SEMA6A expression was low in resting LCs generated in vitro and was enhanced by interferon (IFN)-gamma but not by interleukin-4, interleukin-10, IFN-alpha/beta, or lipopolysaccharide. Most IFN-gamma-induced SEMA6A-positive cells remained immature with low CD83 and DC-LAMP/CD208 expression, but they expressed CCR7 and responded to macrophage inflammatory protein-3beta (MIP-3beta/CCL19). The expression of SEMA6A, for which the ligand and function remain unknown, may therefore identify an alternative IFN-gamma-dependent activation status of LCs in vivo.


Assuntos
Histiocitose/metabolismo , Interferon gama/farmacologia , Células de Langerhans/metabolismo , Linfadenite/metabolismo , Semaforinas/metabolismo , Adulto , Animais , Anticorpos Monoclonais , Antígenos CD , Osso e Ossos/imunologia , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Encéfalo/metabolismo , Movimento Celular , Quimiocina CCL19 , Quimiocinas CC/farmacologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Epitélio/imunologia , Epitélio/metabolismo , Epitélio/patologia , Histiocitose/patologia , Humanos , Imunoglobulinas , Interferon-alfa/farmacologia , Interferon beta/farmacologia , Interleucina-10/farmacologia , Interleucina-4/farmacologia , Células de Langerhans/imunologia , Lipopolissacarídeos/farmacologia , Linfonodos/citologia , Linfonodos/imunologia , Linfonodos/patologia , Linfadenite/patologia , Proteínas Inflamatórias de Macrófagos , Glicoproteínas de Membrana , Camundongos , Camundongos Endogâmicos BALB C , Tonsila Palatina/imunologia , Tonsila Palatina/metabolismo , Tonsila Palatina/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CCR7 , Receptores de Quimiocinas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Semaforinas/genética , Semaforinas/imunologia , Pele/imunologia , Pele/metabolismo , Pele/patologia
13.
Clin Cancer Res ; 10(22): 7466-74, 2004 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-15569976

RESUMO

PURPOSE: Although dendritic cells (DC) and T cells can infiltrate primary breast carcinoma, it remains unclear whether the immune response influences the clinical outcome. EXPERIMENTAL DESIGN: T lymphocytes and DC infiltration within primary tumors was investigated in 152 patients with invasive nonmetastatic breast cancer. CD1a, CD3, CD68, CD123, CD207/Langerin, and CD208/DC-LAMP expression was assessed with semiquantitative immunohistochemical analysis. Expression of chemokines involved in DC migration (MIP-3a/CCL20, MIP-3b/CCL19, and 6Ckine/CCL21) was also examined. The correlation between these markers and the characteristics of the tumors, as well as relapse-free and overall survival was analyzed. Significant prognostic parameters were then tested in a validation series. RESULTS: Infiltration by immature CD207/Langerin+ DC was found in a third of the cancers and did not correlate with clinicopathological data. Presence of mature CD208/DC-LAMP+ DC (56%) and CD3+ T cells (82%) strongly correlated with lymph node involvement and tumor grade. Among the chemokines analyzed, only the presence of MIP-3b/CCL19 in 57% of the tumors correlated with prolonged overall survival. CD123+ plasmacytoid DC (pDC) infiltrated 13% of the primary tumors. Their presence was strongly associated with shorter overall survival (93% versus 58% at 60 months) and relapse-free survival (90% versus 37% at 60 months) and was found to be an independent prognostic factor for overall survival and relapse-free survival and confirmed in an independent validation series of 103 patients. CONCLUSIONS: Infiltration by pDC of primary localized breast tumor correlates with an adverse outcome, suggesting their contribution in the progression of breast cancer.


Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Células Dendríticas/citologia , Células Dendríticas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD , Antígenos de Superfície/biossíntese , Complexo CD3/biossíntese , Progressão da Doença , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Subunidade alfa de Receptor de Interleucina-3 , Lectinas Tipo C/biossíntese , Metástase Linfática , Lectinas de Ligação a Manose/biossíntese , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Receptores de Quimiocinas/biossíntese , Receptores de Interleucina-3/biossíntese , Recidiva , Linfócitos T/citologia , Fatores de Tempo , Resultado do Tratamento
14.
Am J Pathol ; 164(3): 861-71, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14982840

RESUMO

Dendritic cell-lysosomal associated membrane protein (DC-LAMP)/CD208, a member of the lysosomal associated membrane protein (LAMP) family, is specifically expressed by human DCs on activation. However, its mouse counterpart could not be detected in mature DCs. The present study demonstrates that DC-LAMP is constitutively expressed by mouse, sheep, and human type II pneumocytes. Confocal and immunoelectron microscopy showed that mouse DC-LAMP protein co-localizes with lbm180, a specific marker for the limiting membrane of lamellar bodies that contain surfactant protein B, as well as with intracellular MHC class II molecules that accumulate in the same organelles. Expression of DC-LAMP was also occasionally detected at the cell surface of type II pneumocytes. Interestingly, human bronchioloalveolar carcinoma tumor cells, which correspond to transformed type II pneumocytes, express DC-LAMP. Similar observations were made in the Jaagsiekte sheep retrovirus-associated ovine pulmonary adenocarcinoma, a model of human bronchioloalveolar carcinoma. This study establishes that DC-LAMP is constitutively expressed in normal type II pneumocytes. Furthermore, DC-LAMP appears to be a marker of transformed type II pneumocytes as well, an observation that may help the study and the classification of human lung adenocarcinomas.


Assuntos
Antígenos CD/biossíntese , Biomarcadores Tumorais/análise , Células Dendríticas/imunologia , Células Dendríticas/ultraestrutura , Pulmão/citologia , Adenocarcinoma Bronquioloalveolar/metabolismo , Adenocarcinoma Bronquioloalveolar/patologia , Animais , Antígenos CD/ultraestrutura , Northern Blotting , Transformação Celular Neoplásica , Células Cultivadas , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Pulmão/ultraestrutura , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Glicoproteínas de Membrana Associadas ao Lisossomo , Camundongos , Microscopia Confocal , Microscopia Imunoeletrônica , Especificidade da Espécie
15.
J Immunol ; 171(12): 6466-77, 2003 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-14662846

RESUMO

We report in this study the generation of a novel rat mAb that recognizes mouse plasmacytoid dendritic cells (pDC). This Ab, named 120G8, stains a small subset of CD11c(low) spleen cell with high specificity. This population produces high amounts of IFN-alpha upon in vitro viral stimulation. Both ex vivo- and in vitro-derived 120G8(+) cells display a phenotype identical with that of the previously described mouse pDC (B220(high)Ly6C(high)Gr1(low)CD11b(-)CD11c(low)). Mice treated with 120G8 mAb are depleted of B220(high)Ly6C(high)CD11c(low) cells and have a much-reduced ability to produce IFN-alpha in response to in vivo CpG stimulation. The mAb 120G8 stains all and only B220(high)Ly6C(high)CD11c(low) pDC in all lymphoid organs. Immunohistochemical studies performed with this mAb indicate that pDC are located in the T cell area of spleen, lymph nodes, and Peyer's patches. Although the Ag recognized by 120G8 is not yet known, we show that its expression is up-regulated by type I IFN on B cells and DC. Using this mAb in immunofluorescence studies demonstrates strain- and organ-specific differences in the frequency of pDC and other DC subsets. 129Sv mice have a much higher frequency of pDC, together with a lower frequency of conventional CD8alpha(+)CD11c(high) DC, compared with C57BL/6 mice, both in spleen and blood. The higher ability of 129Sv mice to produce IFN-alpha in vivo is related to a higher number of pDC, but also to a higher ability of pDC from 129Sv mice to produce IFN-alpha in vitro in response to viral stimulation.


Assuntos
Anticorpos Monoclonais/metabolismo , Células Dendríticas/citologia , Células Dendríticas/imunologia , Animais , Anticorpos Bloqueadores/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/biossíntese , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Antígeno CD11c/biossíntese , Diferenciação Celular/imunologia , Células Cultivadas , Células Dendríticas/química , Células Dendríticas/metabolismo , Feminino , Imuno-Histoquímica , Imunofenotipagem , Interferon Tipo I/farmacologia , Interferon gama/antagonistas & inibidores , Interferon gama/biossíntese , Antígenos Comuns de Leucócito/biossíntese , Contagem de Leucócitos , Leucopenia/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Endogâmicos DBA , Camundongos Nus , Camundongos SCID , Orthomyxoviridae/imunologia , Ratos , Especificidade da Espécie , Baço/citologia , Baço/imunologia , Baço/metabolismo , Coloração e Rotulagem , Regulação para Cima/imunologia
16.
Eur J Immunol ; 33(9): 2619-29, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12938238

RESUMO

DC-LAMP, a member of the lysosomal-associated membrane protein (LAMP) family, is specifically expressed by human dendritic cells (DC) upon activation and therefore serves as marker of human DC maturation. DC-LAMP is detected first in activated human DC within MHC class II molecules-containing compartments just before the translocation of MHC class II-peptide complexes to the cell surface, suggesting a possible involvement in this process. The present study describes the cloning and characterization of mouse DC-LAMP, whose predicted protein sequence is over 50% identical to the human counterpart. The mouse DC-LAMP gene spans over 25 kb and shares syntenic chromosomal localization (16B2-B4 and 3q26) and conserved organization with the human DC-LAMP gene. Analysis of mouse DC-LAMP mRNA and protein revealed the expression in lung peripheral cells, but also its unexpected absence from mouse lymphoid organs and from mouse DC activated either in vitro or in vivo. In conclusion, mouse DC-LAMP is not a marker of mature mouse DC and this observation raises new questions regarding the role of human DC-LAMP in human DC.


Assuntos
Antígenos CD/genética , Clonagem Molecular , Células Dendríticas/metabolismo , Animais , Antígenos CD/metabolismo , Sequência de Bases , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Humanos , Pulmão/metabolismo , Glicoproteínas de Membrana Associadas ao Lisossomo , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos/fisiologia , Filogenia , RNA Mensageiro/metabolismo , Alinhamento de Sequência
17.
J Immunol ; 168(2): 782-92, 2002 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-11777972

RESUMO

Human (h)Langerin/CD207 is a C-type lectin of Langerhans cells (LC) that induces the formation of Birbeck granules (BG). In this study, we have cloned a cDNA-encoding mouse (m)Langerin. The predicted protein is 66% homologous to hLangerin with conservation of its particular features. The organization of human and mouse Langerin genes are similar, consisting of six exons, three of which encode the carbohydrate recognition domain. The mLangerin gene maps to chromosome 6D, syntenic to the human gene on chromosome 2p13. mLangerin protein, detected by a mAb as a 48-kDa species, is abundant in epidermal LC in situ and is down-regulated upon culture. A subset of cells also expresses mLangerin in bone marrow cultures supplemented with TGF-beta. Notably, dendritic cells in thymic medulla are mLangerin-positive. By contrast, only scattered cells express mLangerin in lymph nodes and spleen. mLangerin mRNA is also detected in some nonlymphoid tissues (e.g., lung, liver, and heart). Similarly to hLangerin, a network of BG form upon transfection of mLangerin cDNA into fibroblasts. Interestingly, substitution of a conserved residue (Phe(244) to Leu) within the carbohydrate recognition domain transforms the BG in transfectant cells into structures resembling cored tubules, previously described in mouse LC. Our findings should facilitate further characterization of mouse LC, and provide insight into a plasticity of dendritic cell organelles which may have important functional consequences.


Assuntos
Antígenos de Superfície/isolamento & purificação , Células Dendríticas/química , Células de Langerhans/química , Tecido Linfoide/química , Lectinas de Ligação a Manose , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Anticorpos Monoclonais/química , Antígenos CD/biossíntese , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos CD/isolamento & purificação , Antígenos de Superfície/biossíntese , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Sequência de Bases , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linhagem Celular , Células Cultivadas , Meios de Cultura/farmacologia , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/metabolismo , DNA Complementar/isolamento & purificação , Células Dendríticas/imunologia , Humanos , Células de Langerhans/imunologia , Lectinas/biossíntese , Lectinas/genética , Lectinas/imunologia , Lectinas/isolamento & purificação , Lectinas Tipo C , Leucina/genética , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microtúbulos/genética , Microtúbulos/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Fenilalanina/genética , RNA Mensageiro/metabolismo , Transfecção , Fator de Crescimento Transformador beta/farmacologia
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