RESUMO
Taurine (Tau) is a special sulphur-containing amino acid and has been widely used as a dietary supplement. Although Tau exists in lymphocytes in large quantities, the physiological significance of Tau to modulate human immunity is unknown. In the present study, we first found that Tau regulates the B-cell receptor (BCR)-mediated signal transduction and induces the B cells activation. The IgG production of mice after ovalbumin immunization was also increased by Tau administration. Moreover, the isothermal titration calorimetry and surface plasmon resonance analysis have shown that Tau specifically bound to the IgG2a-BCR. The Tau could bind to IgG F(ab')2 regions via fluorescence spectroscopy analysis. In the molecular docking analysis, Tau bound to the framework regions (FRs) of variable region of the heavy chains (VH ) and in the light chains (VL ) of IgG2a-BCR. Our results suggested that Tau could improve the activation of B cells by interaction with the VH /VL FRs of BCR.
Assuntos
Cadeias Pesadas de Imunoglobulinas , Região Variável de Imunoglobulina , Animais , Camundongos , Humanos , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/metabolismo , Taurina , Simulação de Acoplamento Molecular , Receptores de Antígenos de Linfócitos B , Imunoglobulina GRESUMO
The diversity of three hypervariable loops in antibody heavy chain and light chain, termed the complementarity-determining regions (CDRs), defines antibody's binding affinity and specificity owing to the direct contact between the CDRs and antigens. These CDR regions typically contain tyrosine (Tyr) residues that are known to engage in both nonpolar and pi stacking interaction with antigens through their complementary aromatic ring side chains. Nearly two decades ago, sulfotyrosine residue (sTyr), a negatively charged Tyr formed by Golgi-localized membrane-bound tyrosylprotein sulfotransferases during protein trafficking, were also found in the CDR regions and shown to play an important role in modulating antibody-antigen interaction. This breakthrough finding demonstrated that antibody repertoire could be further diversified through post-translational modifications, in addition to the conventional genetic recombination. This review article summarizes the current advances in the understanding of the Tyr-sulfation modification mechanism and its application in potentiating protein-protein interaction for antibody engineering and production. Challenges and opportunities are also discussed.
Assuntos
Regiões Determinantes de Complementaridade , Cadeias Pesadas de Imunoglobulinas , Regiões Determinantes de Complementaridade/genética , Cadeias Pesadas de Imunoglobulinas/genética , Antígenos , Complexo de Golgi/metabolismo , Tirosina/metabolismoRESUMO
We aimed at molecularly dissecting the anatomical heterogeneity of small lymphocytic lymphoma (SLL), by analysing a cohort of 12 patients for whom paired DNA from a lymph node biopsy and circulating cells, as well as plasma-circulating tumour DNA (ctDNA) was available. Notably, the analyses of the lymph node biopsy and of circulating cells complement each other since a fraction of mutations (20·4% and 36·4%, respectively) are unique to each compartment. Plasma ctDNA identified two additional unique mutations. Consistently, the different synchronous sources of tumour DNA complement each other in informing on driver gene mutations in SLL harbouring potential prognostic and/or predictive value.
Assuntos
Aberrações Cromossômicas , DNA de Neoplasias/sangue , Leucemia Linfocítica Crônica de Células B/patologia , Linfonodos/patologia , Adenina/análogos & derivados , Adenina/uso terapêutico , Idoso , Biópsia , Deleção Cromossômica , Cromossomos Humanos Par 12 , Cromossomos Humanos Par 13/ultraestrutura , Cromossomos Humanos Par 17/ultraestrutura , Variações do Número de Cópias de DNA , DNA de Neoplasias/análise , Feminino , Genes de Imunoglobulinas , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Imunoterapia , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/genética , Linfonodos/química , Masculino , Pessoa de Meia-Idade , Mutação , Piperidinas/uso terapêuticoRESUMO
Camels belong to a group of animals, where the structure of placenta does not allow intrauterine transfer of maternal immunoglobulins to the fetus and maternal immunity is exclusively transferred by colostrum to the newborn calf. There are few studies on the passive transfer of maternal immunity in the dromedary camel. This study determined total immunoglobulin G concentration, heavy chain antibody (HCAbs) levels, and neutrophils to lymphocytes ratio (NLR) in female camels and their newborn calves. For this, samples were collected from nine she-camels (blood and colostrum) and their calves (blood). IgG concentration and HCAb level were determined in mother serum and colostrum as well as in calf serum using ELISA. The NLR was calculated after the estimation of relative fractions of neutrophils and lymphocytes in collected blood samples using a blood cell analyzer. Both IgG and HCAbs were higher concentrated in camel colostrum than in mother serum. At parturition and before the first colostrum intake, calf serum did not contain any measurable concentration of IgG and only low levels of HCAbs. After colostrum consumption, a rise in IgG and HCAb levels was observed in calf serum. For total IgG, a minimum was reached on day 30 postnatum. While a significant increase in IgG concentration was seen on day 60 postnatum, no significant rise was measured in HCAbs at that age. Only post-colostrum IgG levels in calf serum correlated positively with IgG levels in mother colostrum. Directly after birth, newborn calves showed significantly higher NLR than their mothers. This indicates a pro-inflammatory nature of the calf immune response. The decrease of the NLR on day 60 postnatum may argue for the maturation of the calf immune response at this age.
Assuntos
Camelus/imunologia , Imunoglobulina G/sangue , Cadeias Pesadas de Imunoglobulinas/sangue , Linfócitos/imunologia , Neutrófilos/imunologia , Animais , Animais Recém-Nascidos/imunologia , Colostro/imunologia , FemininoRESUMO
Systemic TNF neutralization can be used as a therapy for several autoimmune diseases. To evaluate the effects of cell type-restricted TNF blockade, we previously generated bispecific antibodies that can limit TNF secretion by myeloid cells (myeloid cell-specific TNF inhibitors or MYSTIs). In this study several such variable domain (VH) of a camelid heavy-chain only antibody-based TNF inhibitors were compared in relevant experimental models, both in vitro and in vivo. Pretreatment with MYSTI-2, containing the anti-F4/80 module, can restrict the release of human TNF (hTNF) from LPS-activated bone marrow-derived macrophage (BMDM) cultures of humanized TNF knock-in (mice; hTNFKI) more effectively than MYSTI-3, containing the anti-CD11b module. MYSTI-2 was also superior to MYSTI-3 in providing in vivo protection in acute toxicity model. Finally, MYSTI-2 was at least as effective as Infliximab in preventing collagen antibody-induced arthritis. This study demonstrates that a 33 kDa bispecific mini-antibody that specifically restricts TNF secretion by macrophages is efficient for amelioration of experimental arthritis.
Assuntos
Anticorpos Monoclonais/farmacologia , Artrite Experimental/terapia , Antígeno CD11b/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Cadeias Pesadas de Imunoglobulinas/farmacologia , Células Progenitoras Mieloides/efeitos dos fármacos , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Inibidores do Fator de Necrose Tumoral/farmacologia , Animais , Antirreumáticos/farmacologia , Artrite Experimental/genética , Artrite Experimental/imunologia , Artrite Experimental/patologia , Antígeno CD11b/genética , Antígeno CD11b/imunologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Expressão Gênica , Humanos , Infliximab/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Transgênicos , Células Progenitoras Mieloides/imunologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The mechanism of action of recombinant IgG2/4 antibodies involves blocking of their target without the induction of effector functions. Examples are eculizumab (Soliris®), which is used clinically to block complement factor C5, as well as anti-human CD14 (r18D11) and anti-porcine CD14 (rMIL2) produced in our laboratory. So far, no proper IgG2/4 control antibody has been available for controlled validation of IgG2/4 antibody functions. Here, we describe the design of a recombinant control antibody (NHDL), which was generated by combining the variable light (VL) and heavy (VH) chains from two unrelated specificities. NHDL was readily expressed and purified as a stable IgG2/4 antibody, and showed no detectable specificity toward any putative antigen present in human or porcine blood. The approach of artificial VL/VH combination may be adopted for the design of other recombinant control antibodies.
Assuntos
Anticorpos Monoclonais/genética , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Proteínas Recombinantes de Fusão/genética , Animais , Anticorpos Monoclonais/metabolismo , Terapia Biológica , Proteínas Sanguíneas/metabolismo , Epitopos/metabolismo , Humanos , Receptores de Lipopolissacarídeos/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Camundongos , Placebos , Engenharia de Proteínas , SuínosRESUMO
The expression levels of immunoglobulin elements and their receptors are important markers for health and disease. Within the immunoglobulin locus, the constant regions and the variable region families are associated with certain pathologies, yet a holistic view of the interaction between the expressions of the multiple genes remain to be fully characterized. There is thus an important need to quantify antibody elements, their receptors and the receptor subunits in blood (PBMC cDNA) for both screening and detailed studies of such associations. Leveraging on qPCR, we designed primers for all Vκ1-6, VH1-7, Vλ1-11, nine CH isotypes, Cκ, Cκ, Cλ1 &3, FcεRI α,ß, and γ subunits, all three FcγR and their subunits, and FcαR. Validating this on a volunteer PBMC cDNA, we report a qPCR primer set repertoire that can quantify the relative expression of all the above genes to the GAPDH housekeeping gene, with implications and uses in both clinical monitoring and research.
Assuntos
Primers do DNA , Sistema Imunitário/fisiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Receptores Fc/genética , DNA Complementar , Expressão Gênica , Humanos , Cadeias Pesadas de Imunoglobulinas/análise , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/análise , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/análise , Região Variável de Imunoglobulina/genética , Leucócitos Mononucleares , RNA Mensageiro/análise , Receptores Fc/análiseRESUMO
Antigen receptor assembly in lymphocytes involves stringently-regulated coordination of specific DNA rearrangement events across several large chromosomal domains. Previous studies indicate that transcription factors such as paired box 5 (PAX5), Yin Yang 1 (YY1), and CCCTC-binding factor (CTCF) play a role in regulating the accessibility of the antigen receptor loci to the V(D)J recombinase, which is required for these rearrangements. To gain clues about the role of CTCF binding at the murine immunoglobulin heavy chain (IgH) locus, we utilized a computational approach that identified 144 putative CTCF-binding sites within this locus. We found that these CTCF sites share a consensus motif distinct from other CTCF sites in the mouse genome. Additionally, we could divide these CTCF sites into three categories: intergenic sites remote from any coding element, upstream sites present within 8 kb of the VH-leader exon, and recombination signal sequence (RSS)-associated sites characteristically located at a fixed distance (â¼18 bp) downstream of the RSS. We noted that the intergenic and upstream sites are located in the distal portion of the VH locus, whereas the RSS-associated sites are located in the DH-proximal region. Computational analysis indicated that the prevalence of CTCF-binding sites at the IgH locus is evolutionarily conserved. In all species analyzed, these sites exhibit a striking strand-orientation bias, with >98% of the murine sites being present in one orientation with respect to VH gene transcription. Electrophoretic mobility shift and enhancer-blocking assays and ChIP-chip analysis confirmed CTCF binding to these sites both in vitro and in vivo.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Cadeias Pesadas de Imunoglobulinas/genética , Imunidade Adaptativa/genética , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Rearranjo Gênico , Humanos , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina , Células K562 , Camundongos , Camundongos Knockout , Células NIH 3T3 , Motivos de Nucleotídeos , Sequências Reguladoras de Ácido Nucleico , Proteínas Repressoras/metabolismoRESUMO
Nuclear hormone receptors including the estrogen receptor (ERα) and the retinoic acid receptor regulate a plethora of biological functions including reproduction, circulation and immunity. To understand how estrogen and other nuclear hormones influence antibody production, we characterized total serum antibody isotypes in female and male mice of C57BL/6J, BALB/cJ and C3H/HeJ mouse strains. Antibody levels were higher in females compared to males in all strains and there was a female preference for IgG2b production. Sex-biased patterns were influenced by vitamin levels, and by antigen specificity toward influenza virus or pneumococcus antigens. To help explain sex biases, we examined the direct effects of estrogen on immunoglobulin heavy chain sterile transcript production among purified, lipopolysaccharide-stimulated B cells. Supplemental estrogen in B-cell cultures significantly increased immunoglobulin heavy chain sterile transcripts. Chromatin immunoprecipitation analyses of activated B cells identified significant ERα binding to estrogen response elements (EREs) centered within enhancer elements of the immunoglobulin heavy chain locus, including the Eµ enhancer and hypersensitive site 1,2 (HS1,2) in the 3' regulatory region. The ERE in HS1,2 was conserved across animal species, and in humans marked a site of polymorphism associated with the estrogen-augmented autoimmune disease, lupus. Taken together, the results highlight: (i) the important targets of ERα in regulatory regions of the immunoglobulin heavy chain locus that influence antibody production, and (ii) the complexity of mechanisms by which estrogen instructs sex-biased antibody production profiles.
Assuntos
Formação de Anticorpos/genética , Elementos Facilitadores Genéticos , Cadeias Pesadas de Imunoglobulinas/genética , Receptores de Estrogênio/metabolismo , Elementos de Resposta/genética , Caracteres Sexuais , Animais , Formação de Anticorpos/imunologia , Sítios de Ligação , Cadeias Pesadas de Imunoglobulinas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Elementos de Resposta/imunologiaRESUMO
Receptor tyrosine kinase-like orphan receptor 2 (ROR2) has been identified as a highly relevant tumor-associated antigen in a variety of cancer indications of high unmet medical need, including renal cell carcinoma and osteosarcoma, making it an attractive target for targeted cancer therapy. Here, we describe the de novo discovery of fully human ROR2-specific antibodies and potent antibody drug conjugates (ADCs) derived thereof by combining antibody discovery from immune libraries of human immunoglobulin transgenic animals using the Transpo-mAb mammalian cell-based IgG display platform with functional screening for internalizing antibodies using a secondary ADC assay. The discovery strategy entailed immunization of transgenic mice with the cancer antigen ROR2, harboring transgenic IgH and IgL chain gene loci with limited number of fully human V, D, and J gene segments. This was followed by recovering antibody repertoires from the immunized animals, expressing and screening them as full-length human IgG libraries by transposon-mediated display in progenitor B lymphocytes ("Transpo-mAb Display") for ROR2 binding. Individual cellular "Transpo-mAb" clones isolated by single cell sorting and capable of expressing membrane-bound as well as secreted human IgG were directly screened during antibody discovery, not only for high affinity binding to human ROR2, but also functionally as ADCs using a cytotoxicity assay with a secondary anti-human IgG-toxin-conjugate. Using this strategy, we identified and validated 12 fully human, monoclonal anti-human ROR2 antibodies with nanomolar affinities that are highly potent as ADCs and could be promising candidates for the therapy of human cancer. The screening for functional and internalizing antibodies during the early phase of antibody discovery demonstrates the utility of the mammalian cell-based Transpo-mAb Display platform to select for functional binders and as a powerful tool to improve the efficiency for the development of therapeutically relevant ADCs.
Assuntos
Anticorpos Monoclonais Humanizados/isolamento & purificação , Anticorpos Monoclonais/isolamento & purificação , Imunoconjugados/isolamento & purificação , Neoplasias/terapia , Células Precursoras de Linfócitos B/fisiologia , Animais , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Humanos , Imunização , Imunoconjugados/farmacologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Imunotoxinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/imunologia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/imunologia , Análise de Célula Única , Éxons VDJ/genéticaRESUMO
Monoclonal antibodies are playing an increasing role in both human and animal health. Different strategies of protein and chemical engineering, including humanization techniques of non-human antibodies were applied successfully to optimize clinical performances of antibodies. Despite the emergence of techniques allowing the development of fully human antibodies such as transgenic Xeno-mice, antibody humanization remains a standard procedure for therapeutic antibodies. An important prerequisite for antibody humanization requires standardized numbering methods to define precisely complementary determining regions (CDR), frameworks and residues from the light and heavy chains that affect the binding affinity and/or specificity of the antibody-antigen interaction. The recently generated deep-sequencing data and the increasing number of solved three-dimensional structures of antibodies from human and non-human origins have led to the emergence of numerous databases. However, these different databases use different numbering conventions and CDR definitions. In addition, the large fluctuation of the variable chain lengths, especially in CDR3 of heavy chains (CDRH3), hardly complicates the comparison and analysis of antibody sequences and the identification of the antigen binding residues. This review compares and discusses the different numbering schemes and "CDR" definition that were established up to date. Furthermore, it summarizes concepts and strategies used for numbering residues of antibodies and CDR residues identification. Finally, it discusses the importance of specific sets of residues in the binding affinity and/or specificity of immunoglobulins.
Assuntos
Aminoácidos/imunologia , Anticorpos Monoclonais/imunologia , Regiões Determinantes de Complementaridade/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/genética , Camundongos , Domínios ProteicosRESUMO
Cancer patients receiving chemotherapy often experience taste and smell abnormalities (TSA). To date, the underlying molecular mechanisms of this frequent side-effect have not been determined and effective treatments are not available. This study assessed the feasibility of lactoferrin (LF) supplementation as a treatment for TSA and investigate the related mechanisms through salivary proteome analysis. Nineteen cancer patients with established TSA following chemotherapy administration were enrolled in this study. Cancer patients and additional 12 healthy subjects took LF supplements, 3 tablets per day (250 mg per tablet), for 30 days. Saliva was collected at three timepoints: baseline, 30-day LF supplementation, and 30-day post-LF supplementation. Patient's TSA level, salivary proteome, and salivary minerals at each LF treatment stage were analyzed. High TSA level was associated with high concentration of salivary Fe and loss of critical salivary immune proteins. LF supplementation significantly decreased the concentration of salivary Fe (P = 0.025), increased the abundance (P < 0.05) of salivary α-amylase and Zn-α-2-GP, and led to an overall increase of expression (≥2-fold changes) of immune proteins including immunoglobulin heavy chain, annexin A1, and proteinase inhibitor. Abundance of α-amylase and SPLUNC2 were further increased (P < 0.05) at 30-day post-LF supplementation in cancer patients. At the same time, total TSA score was significantly reduced (P < 0.001) in chemotherapy patients. This study demonstrated the feasibility of developing lactoferrin supplementation as a treatment to reduce TSA caused by chemotherapy and improve cancer patient's oral immunity.
Assuntos
Antineoplásicos/efeitos adversos , Suplementos Nutricionais , Lactoferrina/uso terapêutico , Transtornos do Olfato/terapia , Saliva/metabolismo , Proteínas e Peptídeos Salivares/metabolismo , Distúrbios do Paladar/terapia , Idoso , Antioxidantes/efeitos adversos , Antioxidantes/uso terapêutico , Biomarcadores/metabolismo , Suplementos Nutricionais/efeitos adversos , Estudos de Viabilidade , Feminino , Humanos , Imunidade nas Mucosas/efeitos dos fármacos , Cadeias Pesadas de Imunoglobulinas/metabolismo , Ferro/metabolismo , Lactoferrina/efeitos adversos , Masculino , Pessoa de Meia-Idade , Minerais/metabolismo , Transtornos do Olfato/induzido quimicamente , Transtornos do Olfato/metabolismo , Transtornos do Olfato/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , Proteômica/métodos , Saliva/enzimologia , Saliva/imunologia , Eliminação Salivar/efeitos dos fármacos , Autorrelato , Índice de Gravidade de Doença , Distúrbios do Paladar/induzido quimicamente , Distúrbios do Paladar/metabolismo , Distúrbios do Paladar/fisiopatologiaRESUMO
B-1 cells are a unique subset of B cells that are positively selected for expressing autoreactive BCRs. We isolated RNA from peritoneal (B-1a, B-1b, B-2) and splenic (B-1a, marginal zone, follicular) B cells from C57BL/6 mice and used 5'-RACE to amplify the IgH V region using massively parallel sequencing. By analyzing 379,000 functional transcripts, we demonstrate that B-1a cells use a distinct and restricted repertoire. All B-1 cell subsets, especially peritoneal B-1a cells, had a high proportion of sequences without N additions, suggesting predominantly prenatal development. Their transcripts differed markedly and uniquely contained VH11 and VH12 genes, which were rearranged only with a restricted selection of D and J genes, unlike other V genes. Compared to peritoneal B-1a, the peritoneal B-1b repertoire was larger, had little overlap with B-1a, and most sequences contained N additions. Similarly, the splenic B-1a repertoire differed from peritoneal B-1a sequences, having more unique sequences and more frequent N additions, suggesting influx of B-1a cells into the spleen from nonperitoneal sites. Two CDR3s, previously described as Abs to bromelain-treated RBCs, comprised 43% of peritoneal B-1a sequences. We show that a single-chain variable fragment designed after the most prevalent B-1a sequence bound oxidation-specific epitopes such as the phosphocholine of oxidized phospholipids. In summary, we provide the IgH V region library of six murine B cell subsets, including, to our knowledge for the first time, a comparison between B-1a and B-1b cells, and we highlight qualities of B-1 cell Abs that indicate unique selection processes.
Assuntos
Anticorpos/genética , Anticorpos/imunologia , Subpopulações de Linfócitos B/imunologia , Baço/imunologia , Sequência de Aminoácidos , Animais , Diversidade de Anticorpos/genética , Diversidade de Anticorpos/imunologia , Sequência de Bases , Feminino , Genes de Imunoglobulinas/genética , Genes de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Species A Rotaviruses (RVA) remain a leading cause of mortality in children under 5 years of age. Current treatment options are limited. We assessed the efficacy of two VP6-specific llama-derived heavy chain antibody fragments (VHH) -2KD1 and 3B2- as an oral prophylactic and therapeutic treatment against RVA-induced diarrhea in a neonatal mouse model inoculated with virulent murine RVA (ECw, G16P[16]I7). Joint therapeutic administration of 2KD1+3B2 (200 µg/dose) successfully reduced diarrhea duration, RVA infection severity and virus shedding in feces. While the same dose of 2KD1 or 3B2 (200 µg) significantly reduced duration of RVA-induced diarrhea, 2KD1 was more effective in diminishing the severity of intestinal infection and RVA shedding in feces, perhaps because 2KD1 presented higher binding affinity for RVA particles than 3B2. Neither prophylactic nor therapeutic administration of the VHH interfered with the host's humoral immune response against RVA. When 2KD1 (200 µg) was administered after diarrhea development, it also significantly reduced RVA intestinal infection and fecal shedding. Host antibody responses against the oral VHH treatment were not detected, nor did viral escape mutants. Our findings show that oral administration of anti-VP6 VHH constitute, not only an effective prophylactic treatment against RVA-associated diarrhea, but also a safe therapeutic tool against RVA infection, even once diarrhea is present. Anti-VP6 VHH could be used complementary to ongoing vaccination, especially in populations that have shown lower immunization efficacy. These VHH could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.
Assuntos
Cadeias Pesadas de Imunoglobulinas/uso terapêutico , Região Variável de Imunoglobulina/uso terapêutico , Infecções por Rotavirus/tratamento farmacológico , Infecções por Rotavirus/virologia , Rotavirus/fisiologia , Animais , Animais Recém-Nascidos , Camelídeos Americanos , Diarreia/tratamento farmacológico , Diarreia/virologia , Fezes/virologia , Concentração de Íons de Hidrogênio , Imunidade Humoral/imunologia , Cadeias Pesadas de Imunoglobulinas/administração & dosagem , Região Variável de Imunoglobulina/administração & dosagem , Intestinos/patologia , Intestinos/virologia , Camundongos Endogâmicos BALB C , Mutação/genética , Filogenia , Proteólise , Infecções por Rotavirus/imunologia , Vírion/metabolismo , Eliminação de Partículas ViraisRESUMO
YY1 has been implicated as a master regulator of germinal center B cell development as YY1 binding sites are frequently present in promoters of germinal center-expressed genes. YY1 is known to be important for other stages of B cell development including the pro-B and pre-B cells stages. To determine if YY1 plays a critical role in germinal center development, we evaluated YY1 expression during B cell development, and used a YY1 conditional knock-out approach for deletion of YY1 in germinal center B cells (CRE driven by the immunoglobulin heavy chain γ1 switch region promoter; γ1-CRE). We found that YY1 is most highly expressed in germinal center B cells and is increased 3 fold in splenic B cells activated by treatment with anti-IgM and anti-CD40. In addition, deletion of the yy1 gene by action of γ1-CRE recombinase resulted in significant loss of GC cells in both un-immunized and immunized contexts with corresponding loss of serum IgG1. Our results show a crucial role for YY1 in the germinal center reaction.
Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Células Precursoras de Linfócitos B/imunologia , Baço/imunologia , Fator de Transcrição YY1/genética , Animais , Anticorpos Anti-Idiotípicos/farmacologia , Anticorpos Monoclonais/farmacologia , Linfócitos B/citologia , Diferenciação Celular , Técnicas de Inativação de Genes , Centro Germinativo/citologia , Imunoglobulina G/sangue , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Integrases/genética , Integrases/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Células Precursoras de Linfócitos B/citologia , Regiões Promotoras Genéticas , Baço/citologia , Fator de Transcrição YY1/deficiência , Fator de Transcrição YY1/imunologiaRESUMO
Conformation of antigen receptor gene loci spatially juxtaposes rearranging gene segments in the appropriate cell lineage and developmental stage. We describe a three-step pathway that establishes the structure of the 2.8-Mb immunoglobulin heavy chain gene (IgH) locus in pro-B cells. Each step uses a different transcription factor and leads to increasing levels of structural organization. CTCF mediates one level of compaction that folds the locus into several 250- to 400-kb subdomains, and Pax5 further compacts the 2-Mb region that encodes variable (VH) gene segments. The 5' and 3' domains are brought together by the transcription factor YY1 to establish the configuration within which gene recombination initiates. Such stepwise mechanisms may apply more generally to establish regulatory fine structure within megabase-sized topologically associated domains.
Assuntos
Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Células Precursoras de Linfócitos B/química , Animais , Fator de Ligação a CCCTC , Células Cultivadas , Hibridização in Situ Fluorescente , Camundongos Endogâmicos C57BL , Fator de Transcrição PAX5/genética , Fator de Transcrição PAX5/metabolismo , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Recombinação Genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , alfa-Amilases Salivares/metabolismo , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismoRESUMO
At their margins, tumors often contain neutrophils, dendritic cells, and activated macrophages, which express class II MHC and CD11b products. The interplay between stromal cells, tumor cells, and migratory cells such as lymphocytes creates opportunities for noninvasive imaging of immune responses. We developed alpaca-derived antibody fragments specific for mouse class II MHC and CD11b products, expressed on the surface of a variety of myeloid cells. We validated these reagents by flow cytometry and two-photon microscopy to obtain images at cellular resolution. To enable noninvasive imaging of the targeted cell populations, we developed a method to site-specifically label VHHs [the variable domain (VH) of a camelid heavy-chain only antibody] with (18)F or (64)Cu. Radiolabeled VHHs rapidly cleared the circulation (t1/2 ≈ 20 min) and clearly visualized lymphoid organs. We used VHHs to explore the possibility of imaging inflammation in both xenogeneic and syngeneic tumor models, which resulted in detection of tumors with remarkable specificity. We also imaged the infiltration of myeloid cells upon injection of complete Freund's adjuvant. Both anti-class II MHC and anti-CD11b VHHs detected inflammation with excellent specificity. Given the ease of manufacture and labeling of VHHs, we believe that this method could transform the manner in which antitumor responses and/or infectious events may be tracked.
Assuntos
Sistema Imunitário/fisiologia , Neoplasias/imunologia , Tomografia por Emissão de Pósitrons , Aminoaciltransferases/fisiologia , Animais , Anticorpos/imunologia , Antineoplásicos/uso terapêutico , Proteínas de Bactérias/fisiologia , Células da Medula Óssea/metabolismo , Radioisótopos de Cobre/química , Cisteína Endopeptidases/fisiologia , Citometria de Fluxo , Radioisótopos de Flúor/química , Adjuvante de Freund , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/patologia , Transplante de Neoplasias , Neoplasias/terapiaRESUMO
The deposition of misfolded peptides and proteins in the form of amyloid fibrils is the hallmark of nearly fifty medical disorders, including Alzheimer's disease, Parkinson's disease, prion diseases and type II diabetes. These disorders, referred to as amyloidoses, generally become apparent late in life. Their psycho-sociological and economic incidence in western societies will be therefore considerable in the coming decades due to the ageing of the population. Neither preventing nor curative treatments are available yet. These disorders constitute therefore a medical challenge of great importance. Thus, an extensive research is being carried out to understand, at the molecular level, (i) how amyloidogenic proteins misfold and convert from their soluble form into amyloid fibrils, and (ii) how these aggregates or some of their oligomeric precursor species are toxic. The formation of amyloid fibrils proceeds through a complex nucleation/polymerisation mechanism with the formation of various species, including small oligomers. In this review, we focus on how VHHs or nanobodies, the antigen-binding domains of camelid heavy-chain antibodies, are being increasingly used to characterise each of the species formed on the pathway of fibril formation in terms of structure, stability, kinetics of formation and toxicity. We first introduce the characteristic features of nanobodies compared to those of conventional antibody fragments. Thereafter, we discuss how nanobodies, due to their unique properties, are used as probes to dissect the molecular mechanisms of misfolding and aggregation of six proteins associated with diseases, i.e. human lysozyme, ß2-microglobulin, α-synuclein, prion, polyadenylate binding protein nuclear 1 and amyloid ß-peptide. A brief general presentation of each disease and the associated peptide/protein is also provided. In addition, we discuss how nanobodies could be used as early diagnostic tools and as novel strategies to treat diseases associated with protein misfolding and aggregation.
Assuntos
Camelídeos Americanos/imunologia , Cadeias Pesadas de Imunoglobulinas/uso terapêutico , Agregação Patológica de Proteínas/tratamento farmacológico , Deficiências na Proteostase/tratamento farmacológico , Anticorpos de Domínio Único/uso terapêutico , Animais , Camelídeos Americanos/genética , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Agregação Patológica de Proteínas/imunologia , Deficiências na Proteostase/imunologia , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologiaRESUMO
In many mammalian species, newborns are agammaglobulinemic; thus, colostrum and milk are the main sources of early protective antibodies. These antibodies are produced in the mother's serum and transferred to mammalian glands a few days before parturition. Here, we have studied the transfer of immunity from a she-camel immunized with human serum albumin (HSA) to her calf via colostrum and milk. Our results show that HSA-specific antibodies are produced in the mother's serum and are subsequently transferred to her colostrum. These specific antibodies are then transferred by suckling to the calf. The calf serum did not contain HSA-reactive antibodies at parturition and before the first feed, after suckling, a rise in reactivity was observed peaking at 24 h postpartum. The involvement of heavy chain antibodies (HCAbs) in the process of immunity transfer was also examined, and it was found that they were also transferred from the colostrum to the calf serum like conventional antibodies.
Assuntos
Camelus/imunologia , Colostro/imunologia , Imunidade Materno-Adquirida , Prenhez , Animais , Animais Lactentes/imunologia , Anticorpos/sangue , Feminino , Imunoglobulina G/sangue , Cadeias Pesadas de Imunoglobulinas/sangue , GravidezRESUMO
A successful HIV-1 vaccine must elicit immune responses that impede mucosal virus transmission, though functional roles of protective HIV-1 Envelope (Env)-specific mucosal antibodies remain unclear. Colostrum is a rich source of readily accessible mucosal B cells that may help define the mucosal antibody response contributing to prevention of postnatal HIV-1 transmission. To examine the HIV-1 Env-specific colostrum B-cell repertoire, single B cells were isolated from 17 chronically HIV-infected, lactating women, producing 51 blood and 39 colostrum HIV-1 Env-specific B-cell antibodies. All HIV-1 Env-specific colostrum-derived antibodies were immunoglobulin (Ig)G1 isotype and had mean heavy chain complementarity-determining region 3 (CDR3) lengths and mutation frequencies similar to those isolated from blood. However, variable heavy chain (VH) gene subfamily 1(â¼)69 usage was higher among colostrum than blood HIV-1 Env-reactive antibodies (49% vs. 20%, P=0.006, Fisher's exact test). Additionally, more HIV-1 Env-specific colostrum antibodies were gp120 specific than those isolated from blood (44% vs. 16%, P=0.005, Fisher's exact test). One cross-compartment HIV-1 Env-specific clonal B-cell lineage was identified. These unique characteristics of colostrum B-cell antibodies suggest selective homing of HIV-1-specific IgG1-secreting memory B cells to the mammary gland and have implications for targeting mucosal B-cell populations by vaccination.