RESUMO
Haptoglobin-related protein (Hpr) is a plasma protein with high sequence similarity to haptoglobin (Hp). Like Hp, Hpr also binds hemoglobin (Hb) with high affinity, but it does not bind to the Hb-Hp receptor CD163 on macrophages. The Hpr concentration is markedly lower than Hp in plasma and its regulation is not understood. In the present study, we have developed non-crossreactive antibodies to Hpr to analyze the Hpr concentration in 112 plasma samples from anonymized individuals and compared it to Hp. The results show that plasma Hpr correlated with Hp concentrations (rho = 0.46, p = .0001). Hpr accounts for on average 0.35% of the Hp/Hpr pool but up to 29% at low Hp levels. Furthermore, the Hpr concentrations were significantly lower in individuals with the Hp2-2 phenotype compared to those with the Hp2-1 or Hp1-1 phenotypes. Experimental binding analysis did not provide evidence that Hpr associates with Hp and in this way is removed via CD163. In conclusion, the Hpr concentration correlates to Hp concentrations and Hp-phenotypes by yet unknown mechanisms independent of CD163-mediated removal of Hb-Hp complexes.
Assuntos
Haptoglobinas , Hemoglobinas , Antígenos de Neoplasias , Proteínas Sanguíneas/genética , Proteínas Cromossômicas não Histona/genética , Haptoglobinas/química , Haptoglobinas/genética , Haptoglobinas/metabolismo , Hemoglobinas/metabolismo , Humanos , FenótipoRESUMO
Haptoglobin (Hp) is an abundant plasma protein scavenging hemoglobin (Hb) via CD163 on macrophages. This process consumes Hp, which therefore negatively correlates to hemolysis. However, exact measurements of Hp plasma levels are complicated by different phenotypes (Hp1-1, Hp2-1, and Hp2-2) forming different oligomeric states with differences in immunoreactivity. In addition, humans have an immune-cross-reactive Hp-related protein. In the present study, we developed Hp-specific monoclonal antibodies for an accurate Hp analysis of the different Hp phenotypes in a panel of 112 anonymous samples from hospitalized individuals subjected to routine Hp immunoturbidimetric measurements. The data revealed immunoturbidimetry as a reliable method in most cases but also that the use of non-phenotype-specific calibrators leads to substantial bias in the measurement of the Hp-concentration, non at least in Hp1-1 individuals. Furthermore, analysis of the Hb-dependence of the CD163 interaction with Hp1-1 and Hp2-2 showed that a higher 'cost-effectiveness' in the consumption of dimeric Hp1-1 versus multimeric Hp phenotypes is a likely contribution to the observed differences in the plasma levels of the Hp phenotypes. In conclusion, the determination of Hp phenotype and the use of phenotype-specific calibrators are essential to obtain a precise estimate of the Hp level in healthy and diseased individuals.
Assuntos
Haptoglobinas , Hemoglobinas , Anticorpos Monoclonais , Proteínas Cromossômicas não Histona/genética , Haptoglobinas/genética , Haptoglobinas/metabolismo , Hemoglobinas/metabolismo , Humanos , FenótipoRESUMO
Variations in the claudin-14 (CLDN14) gene have been linked to increased risk of hypercalciuria and kidney stone formation. However, the exact cellular localization of CLDN14 and its regulation remain to be fully delineated. To this end, we generated a novel antibody that allowed the detection of CLDN14 in paraffin-embedded renal sections. This showed CLDN14 to be detectable in the kidney only after induction of hypercalcemia in rodent models. Protein expression in the kidney is localized exclusively to the thick ascending limbs (TALs), mainly restricted to the cortical and upper medullary portion of the kidney. However, not all cells in the TALs expressed the tight junction protein. In fact, CLDN14 was primarily expressed in cells also expressing CLDN16 but devoid of CLDN10. CLDN14 appeared in very superficial apical cell domains and near cell junctions in a belt-like formation along the apical cell periphery. In transgenic mice, Cldn14 promotor-driven LacZ activity did not show complete colocalization with CLDN14 protein nor was it increased by hypercalcemia, suggesting that LacZ activity cannot be used as a marker for CLDN14 localization and regulation in this model. In conclusion, CLDN14 showed a restricted localization pattern in the apical domain of select cells of the TAL.
Assuntos
Claudinas/metabolismo , Hipercalcemia/metabolismo , Alça do Néfron/metabolismo , Animais , Claudinas/genética , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Hipercalcemia/genética , Hipercalcemia/patologia , Alça do Néfron/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos WistarRESUMO
Functional properties of the paracellular pathway depend critically on the set of claudins (CLDN) expressed at the tight junction. Two syndromes are causally linked to loss-of-function mutations of claudins: hypohidrosis, electrolyte imbalance, lacrimal gland dysfunction, ichthyosis, and xerostomia (HELIX) syndrome caused by genetic variations in the CLDN10 gene and familial hypomagnesemia with hypercalciuria and nephrocalcinosis caused by genetic variations in the CLDN16 or CLDN19 genes. All three genes are expressed in the kidney, particularly in the thick ascending limb (TAL). However, localization of these claudins in humans and rodents remains to be delineated in detail. We studied the segmental and subcellular expression of CLDN10, CLDN16, and CLDN19 in both paraffin-embedded and frozen kidney sections from the adult human, mouse, and rat using immunohistochemistry and immunofluorescence, respectively. Here, CLDN10 was present in a subset of medullary and cortical TAL cells, localizing to basolateral domains and tight junctions in human and rodent kidneys. Weak expression was detected at the tight junction of proximal tubular cells. CLDN16 was primarily expressed in a subset of TAL cells in the cortex and outer stripe of outer medulla, restricted to basolateral domains and tight junctional structures in both human and rodent kidneys. CLDN19 predominantly colocalized with CLDN16 in tight junctions and basolateral domains of the TAL but was also found in basolateral and junctional domains in more distal sites. CLDN10 expression at tight junctions almost never overlapped with that of CLND16 and CLDN19, consistent with distinct junctional pathways with different permeation profiles in both human and rodent kidneys.NEW & NOTEWORTHY This study used immunohistochemistry and immunofluorescence to investigate the distribution of claudin 10, 16, and 19 in the human, mouse, and rat kidney. The findings showed distinct junctional pathways in both human and rodent kidneys, supporting the existence of different permeation profiles in all species investigated.
Assuntos
Claudinas/metabolismo , Túbulos Renais/metabolismo , Animais , Epitélio/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Ratos , Junções Íntimas/metabolismoRESUMO
The epithelial Na+ channel (ENaC) is essential for Na+/K+ homeostasis and blood pressure control. Its activity is regulated by proteases in rodents. To gain more information on proteolytic ENaC regulation in humans, we tested the hypotheses that (1) human kidney α- and γ-ENaC subunits are furin-cleaved, glycosylated, and altered by medication that change plasma aldosterone; (2) prostasin-cleaved γ-ENaC is increased in proteinuria, and (3) cleaved ENaC moieties prevail at the membranes and in urinary extracellular vesicles (uEVs). We developed three monoclonal antibodies (mAbs) targeting (1) the neo-epitope generated after furin cleavage in γ-ENaC (mAb-furin); (2) the intact prostasin cleavage-site in γ-ENaC (mAb-intactRKRK), and (3) the α-ENaC subunit (mAb-alpha). Nephrectomy tissue and uEVs were used for immunoblotting and -histochemistry. In human kidney tissue, mAb-furin detected a ≈ 65-70 kDa protein, compatible with furin-cleaved γ-ENaC; mAb-intactRKRK detected full-length (≈ 90-100 kDa) and furin-cleaved (≈ 70-75 kDa) γ-ENaC. mAb-alpha detected a ≈ 50 kDa protein compatible with furin-cleaved α-subunit. Furin-cleaved γ-ENaC was detected predominantly within membrane fractions and deglycosylation shifted full-length γ-ENaC migration ~ 20 kDa. While γ-ENaC uEV levels were below the detection limit, α-ENaC migrated as intact (≈ 75 kDa) and furin-cleaved (≈ 50 kDa) in uEVs. Kidney levels of α- and γ-ENaC in diuretic- (n = 3) and ACE-inhibitor-treated (n = 4) patients were not different from controls (n = 4). Proteinuric patients (n = 6) displayed similar level of furin-cleaved γ-ENaC as controls (n = 4). Cleaved α-ENaC abundance was significantly lower in the kidneys from proteinuria patients. In conclusion, the study demonstrates ENaC cleavage as an event in human kidney that could contribute to physiological regulation and pathophysiological activation of ENaC.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Epitélio/metabolismo , Furina/metabolismo , Rim/metabolismo , Subunidades Proteicas/metabolismo , Canais de Sódio/metabolismo , Aldosterona/metabolismo , Animais , Diuréticos/farmacologia , Epitélio/efeitos dos fármacos , Glicosilação , Humanos , Rim/efeitos dos fármacos , Camundongos , Proteinúria/metabolismo , Serina Endopeptidases/metabolismo , Sódio/metabolismoRESUMO
Human urinary extracellular vesicles (uEVs) contain proteins from all nephron segments. An assumption for years has been that uEVs might provide a noninvasive liquid biopsy that reflect physiological regulation of transporter protein expression in humans. We hypothesized that protein abundance in human kidney tissue and uEVs are directly related and tested this in paired collections of nephrectomy tissue and urine sample from 12 patients. Kidney tissue was fractioned into total kidney protein, crude membrane (plasma membrane and large intracellular vesicles)-enriched, and intracellular vesicle-enriched fractions as well as sections for immunolabeling. uEVs were isolated from spot urine samples. Antibodies were used to quantify six segment-specific proteins [proximal tubule-expressed Na+-phosphate cotransporters (NaPi-2a), thick ascending limb-expressed Tamm-Horsfall protein and renal outer medullary K+ channels, distal convoluted tubule-expressed NaCl cotransporters, intercalated cell-expressed V-type H+-ATPase subunit G3 (ATP6V1G3), and principal cell-expressed aquaporin 2] and three uEV markers (exosomal CD63, microvesicle marker vesicle-associated membrane protein 3, and ß-actin) in each fraction. By Western blot analysis and immunofluorescence labeling, we found significant positive correlations between the abundance of CD63, NaCl cotransporters, aquaporin 2, and ATP6V1G3, respectively, within the different kidney-derived fractions. We detected all nine proteins in uEVs, but their level did not correlate with kidney tissue protein abundance. uEV protein levels showed higher interpatient variability than kidney-derived fractions, indicating that factors, besides kidney protein abundance, contribute to the uEV protein level. Our data suggest that, in a random sample of nephrectomy patients, uEV protein level is not a predictor of kidney protein abundance.
Assuntos
Células Epiteliais/química , Vesículas Extracelulares/química , Túbulos Renais/química , Proteínas de Membrana Transportadoras/urina , Biomarcadores/urina , Humanos , Túbulos Renais/cirurgia , NefrectomiaRESUMO
Krabbe disease is a severe, fatal neurodegenerative disorder caused by defects in the lysosomal enzyme galactocerebrosidase (GALC). The correct targeting of GALC to the lysosome is essential for the degradation of glycosphingolipids including the primary lipid component of myelin. Over 100 different mutations have been identified in GALC that cause Krabbe disease but the mechanisms by which they cause disease remain unclear. We have generated monoclonal antibodies against full-length human GALC and used these to monitor the trafficking and processing of GALC variants in cell-based assays and by immunofluorescence microscopy. Striking differences in the secretion, processing and endosomal targeting of GALC variants allows the classification of these into distinct categories. A subset of GALC variants are not secreted by cells, not proteolytically processed, and remain trapped in the ER; these are likely to cause disease due to protein misfolding and should be targeted for pharmacological chaperone therapies. Other GALC variants can be correctly secreted by cells and cause disease due to catalytic defects in the enzyme active site, inappropriate post-translational modification or a potential inability to bind essential cofactors. The classification of disease pathogenesis presented here provides a molecular framework for appropriate targeting of future Krabbe disease therapies.
Assuntos
Galactosilceramidase/metabolismo , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/metabolismo , Lisossomos/metabolismo , Mutação/genética , Linhagem Celular , Galactosilceramidase/química , Galactosilceramidase/genética , Humanos , Lisossomos/genética , Processamento de Proteína Pós-TraducionalRESUMO
The vacuolar-type H+-ATPase B1 subunit is heavily expressed in the intercalated cells of the collecting system, where it contributes to H+ transport, but has also been described in other segments of the renal tubule. This study aimed to determine the localization of the B1 subunit of the vacuolar-type H+-ATPase in the early distal nephron, encompassing thick ascending limbs (TAL) and distal convoluted tubules (DCT), in human kidney and determine whether the localization differs between rodents and humans. Antibodies directed against the H+-ATPase B1 subunit were used to determine its localization in paraffin-embedded formalin-fixed mouse, rat, and human kidneys by light microscopy and in sections of Lowicryl-embedded rat kidneys by electron microscopy. Abundant H+-ATPase B1 subunit immunoreactivity was observed in the human kidney. As expected, intercalated cells showed the strongest signal, but significant signal was also observed in apical membrane domains of the distal nephron, including TAL, macula densa, and DCT. In mouse and rat, H+-ATPase B1 subunit expression could also be detected in apical membrane domains of these segments. In rat, electron microscopy revealed that the H+-ATPase B1 subunit was located in the apical membrane. Furthermore, the H+-ATPase B1 subunit colocalized with other H+-ATPase subunits in the TAL and DCT. In conclusion, the B1 subunit is expressed in the early distal nephron. The physiological importance of H+-ATPase expression in these segments remains to be delineated in detail. The phenotype of disease-causing mutations in the B1 subunit may also relate to its presence in the TAL and DCT.
Assuntos
Túbulos Renais Distais/enzimologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Polaridade Celular , Humanos , Imuno-Histoquímica , Túbulos Renais Distais/ultraestrutura , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Especificidade da Espécie , ATPases Vacuolares Próton-Translocadoras/deficiência , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
Significant alterations in maternal calcium (Ca2+) and magnesium (Mg2+) balance occur during lactation. Ca2+ is the primary divalent cation mobilized into breast milk by demineralization of the skeleton and alterations in intestinal and renal Ca2+ transport. Mg2+ is also concentrated in breast milk, but the underlying mechanisms are not well understood. To determine the molecular alterations in Ca2+ and Mg2+ transport in the intestine and kidney during lactation, three groups of female mice consisting of either nonpregnant controls, lactating mice, or mice undergoing involution were examined. The fractional excretion of Ca2+, but not Mg2+, rose significantly during lactation. Renal 1-α hydroxylase and 24-OHase mRNA levels increased markedly, as did plasma 1,25 dihydroxyvitamin D levels. This was accompanied by significant increases in intestinal expression of Trpv6 and S100g in lactating mice. However, no alterations in the expression of cation-permeable claudin-2, claudin-12, or claudins-15 were found in the intestine. In the kidney, increased expression of Trpv5 and Calb1 was observed during lactation, while no changes in claudins involved in Ca2+ and Mg2+ transport (claudin-2, claudin-14, claudin-16, or claudin-19) were found. Consistent with the mRNA expression, expression of both calbindin-D28K and transient receptor potential vanilloid 5 (TRPV5) proteins increased. Colonic Trpm6 expression increased during lactation, while renal Trpm6 remained unaltered. In conclusion, proteins involved in transcellular Ca2+ and Mg2+ transport pathways increase during lactation, while expression of paracellular transport proteins remained unchanged. Increased fractional Ca2+ excretion can be explained by vitamin D-dependent intestinal hyperabsorption and bone demineralization, despite enhanced transcellular Ca2+ uptake by the kidney.
Assuntos
Cálcio/metabolismo , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Rim/metabolismo , Lactação/metabolismo , Magnésio/metabolismo , Glândulas Mamárias Animais/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Adaptação Fisiológica , Animais , Transporte Biológico , Calbindina 1/genética , Calbindina 1/metabolismo , Cálcio/urina , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Claudinas/genética , Claudinas/metabolismo , Feminino , Absorção Intestinal , Mucosa Intestinal/citologia , Rim/citologia , Proteínas de Membrana Transportadoras/genética , Camundongos , Reabsorção Renal , Proteína G de Ligação ao Cálcio S100/genética , Proteína G de Ligação ao Cálcio S100/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Fatores de Tempo , Vitamina D/análogos & derivados , Vitamina D/sangue , Vitamina D3 24-Hidroxilase/genética , Vitamina D3 24-Hidroxilase/metabolismoRESUMO
Surfactant protein D (SP-D) is critical for maintenance of lung homeostasis and provides a first line of defense to pathogens at mucosal surfaces. Polymorphisms in the SP-D-encoding gene SFTPD have been associated with chronic obstructive pulmonary disease and ulcerative colitis. Identification of the immunoreceptors that bind SP-D is essential for understanding its contribution to lung homeostasis and mucosal defense. We located a putative binding motif for the osteoclast-associated receptor (OSCAR) within the SP-D collagenous domain. An OSCAR-Fc fusion protein specifically bound to the collagenous region of recombinant SP-D and captured native SP-D from human bronchoalveolar lavage. OSCAR localized in an intracellular compartment of alveolar macrophages together with SP-D. Moreover, we found OSCAR on the surface of interstitial lung and blood CCR2(+) inflammatory monocytes, which secreted TNF-α when exposed to SP-D in an OSCAR-dependent fashion. OSCAR and SP-D did not exclusively colocalize in lung, as they were also highly expressed in atherosclerotic plaques of human aorta, supporting a role for this interaction in atherosclerosis. Our results identify the OSCAR:SP-D interaction as a potential therapeutic target in chronic inflammatory diseases of the lung as well as other diseases involving tissue accumulation of SP-D, infiltration of inflammatory monocytes, and release of TNF-α.
Assuntos
Inflamação/metabolismo , Monócitos/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , Receptores CCR2/metabolismo , Receptores de Superfície Celular/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Biologia Computacional , Células Espumosas/imunologia , Células Espumosas/metabolismo , Células Espumosas/patologia , Expressão Gênica , Humanos , Inflamação/imunologia , Inflamação/patologia , Espaço Intracelular/metabolismo , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Monócitos/imunologia , Células Mieloides/imunologia , Células Mieloides/metabolismo , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The attenuated yellow fever (YF) vaccine (YF-17D) was developed in the 1930s, yet little is known about the protective mechanisms underlying its efficiency. In this study, we analyzed the relative contribution of cell-mediated and humoral immunity to the vaccine-induced protection in a murine model of YF-17D infection. Using different strains of knockout mice, we found that CD4(+) T cells, B cells, and Abs are required for full clinical protection of vaccinated mice, whereas CD8(+) T cells are dispensable for long-term survival after intracerebral challenge. However, by analyzing the immune response inside the infected CNS, we observed an accelerated T cell influx into the brain after intracerebral challenge of vaccinated mice, and this T cell recruitment correlated with improved virus control in the brain. Using mice deficient in B cells we found that, in the absence of Abs, YF vaccination can still induce some antiviral protection, and in vivo depletion of CD8(+) T cells from these animals revealed a pivotal role for CD8(+) T cells in controlling virus replication in the absence of a humoral response. Finally, we demonstrated that effector CD8(+) T cells also contribute to viral control in the presence of circulating YF-specific Abs. To our knowledge, this is the first time that YF-specific CD8(+) T cells have been demonstrated to possess antiviral activity in vivo.
Assuntos
Anticorpos Antivirais/imunologia , Linfócitos T CD8-Positivos/imunologia , Febre Amarela/imunologia , Vírus da Febre Amarela/imunologia , Imunidade Adaptativa , Animais , Anticorpos Neutralizantes/imunologia , Linfócitos B/imunologia , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/virologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Ligante de CD40/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Quimiotaxia de Leucócito/imunologia , Modelos Animais de Doenças , Feminino , Imunização Passiva , Depleção Linfocítica , Camundongos , Camundongos Knockout , Vacinação , Replicação Viral , Febre Amarela/genética , Febre Amarela/mortalidade , Febre Amarela/prevenção & controle , Vacina contra Febre Amarela/imunologiaRESUMO
We identified a novel, evolutionarily conserved receptor encoded within the human leukocyte receptor complex and syntenic region of mouse chromosome 7, named T cell-interacting, activating receptor on myeloid cells-1 (TARM1). The transmembrane region of TARM1 contained a conserved arginine residue, consistent with association with a signaling adaptor. TARM1 associated with the ITAM adaptor FcRγ but not with DAP10 or DAP12. In healthy mice, TARM1 is constitutively expressed on the cell surface of mature and immature CD11b(+)Gr-1(+) neutrophils within the bone marrow. Following i.p. LPS treatment or systemic bacterial challenge, TARM1 expression was upregulated by neutrophils and inflammatory monocytes and TARM1(+) cells were rapidly recruited to sites of inflammation. TARM1 expression was also upregulated by bone marrow-derived macrophages and dendritic cells following stimulation with TLR agonists in vitro. Ligation of TARM1 receptor in the presence of TLR ligands, such as LPS, enhanced the secretion of proinflammatory cytokines by macrophages and primary mouse neutrophils, whereas TARM1 stimulation alone had no effect. Finally, an immobilized TARM1-Fc fusion protein suppressed CD4(+) T cell activation and proliferation in vitro. These results suggest that a putative T cell ligand can interact with TARM1 receptor, resulting in bidirectional signaling and raising the T cell activation threshold while costimulating the release of proinflammatory cytokines by macrophages and neutrophils.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Citocinas/metabolismo , Macrófagos/imunologia , Neutrófilos/imunologia , Receptores Imunológicos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Granulócitos/imunologia , Granulócitos/metabolismo , Células HEK293 , Antígenos HLA/genética , Humanos , Inflamação/imunologia , Ligantes , Lipopolissacarídeos/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Dados de Sequência Molecular , Neutrófilos/metabolismo , Transporte Proteico/imunologia , Receptores Imunológicos/genética , Proteínas Recombinantes de Fusão/imunologia , Transdução de Sinais/imunologiaRESUMO
C1 inhibitor (C1-INH) is known to form complexes with the lectin complement pathway serine proteases MASP-1 and MASP-2. Deficiency of C1-INH is associated with hereditary angioedema (HAE), an autosomal inherited disease characterized by swelling attacks caused by elevated levels of bradykinin. MASP-1 was shown to cleave high m.w. kininogen into bradykinin; therefore, we hypothesized that MASP-1 levels and the quantity of MASP-1/C1-INH complexes might be associated with different paraclinical and clinical outcomes of HAE. We measured MASP-1 serum concentrations and endogenous MASP-1/C1-INH complex levels in 128 HAE patients and 100 controls. Relatively high levels of pre-existing MASP-1/C1-INH complexes were observed in normal serum, and we found that both the serum levels of MASP-1 and the complex formation between MASP-1 and C1-INH were significantly reduced in HAE patients compared with matched controls (p < 0.0001). The level of MASP-1 and MASP-1/C1-INH complexes in HE patients correlated with the level of C1-INH (p = 0.0009 and p = 0.0047, respectively), the level of C4 (p = 0.0084 and p < 0.0001, respectively), and the number of attacks in the year of blood sampling (p = 0.0075 and p = 0.0058, respectively). In conclusion, we show that MASP-1/C1-INH complexes circulate in normal human blood. The levels of MASP-1 and MASP-1/C1-INH complexes are reduced in HAE patients compared with controls. Both MASP-1 and MASP-1/C1-INH complexes are related to the degree of complement C4 consumption, as well as the severity of disease. These results suggest that MASP-1 may exert a previously unrecognized role in the pathophysiology of HAE.
Assuntos
Angioedemas Hereditários/imunologia , Proteínas Inativadoras do Complemento 1/imunologia , Serina Proteases Associadas a Proteína de Ligação a Manose/imunologia , Complexos Multiproteicos/imunologia , Adulto , Angioedemas Hereditários/sangue , Angioedemas Hereditários/patologia , Proteínas Inativadoras do Complemento 1/metabolismo , Proteína Inibidora do Complemento C1 , Complemento C4/imunologia , Complemento C4/metabolismo , Feminino , Humanos , Masculino , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Pessoa de Meia-Idade , Complexos Multiproteicos/sangue , Índices de Gravidade do TraumaRESUMO
Many genes important in immunity are found as multigene families. The butyrophilin genes are members of the B7 family, playing diverse roles in co-regulation and perhaps in antigen presentation. In humans, a fixed number of butyrophilin genes are found in and around the major histocompatibility complex (MHC), and show striking association with particular autoimmune diseases. In chickens, BG genes encode homologues with somewhat different domain organisation. Only a few BG genes have been characterised, one involved in actin-myosin interaction in the intestinal brush border, and another implicated in resistance to viral diseases. We characterise all BG genes in B12 chickens, finding a multigene family organised as tandem repeats in the BG region outside the MHC, a single gene in the MHC (the BF-BL region), and another single gene on a different chromosome. There is a precise cell and tissue expression for each gene, but overall there are two kinds, those expressed by haemopoietic cells and those expressed in tissues (presumably non-haemopoietic cells), correlating with two different kinds of promoters and 5' untranslated regions (5'UTR). However, the multigene family in the BG region contains many hybrid genes, suggesting recombination and/or deletion as major evolutionary forces. We identify BG genes in the chicken whole genome shotgun sequence, as well as by comparison to other haplotypes by fibre fluorescence in situ hybridisation, confirming dynamic expansion and contraction within the BG region. Thus, the BG genes in chickens are undergoing much more rapid evolution compared to their homologues in mammals, for reasons yet to be understood.
Assuntos
Antígenos de Grupos Sanguíneos/genética , Galinhas/genética , Complexo Principal de Histocompatibilidade/genética , Animais , Sequência de Bases , Butirofilinas , Galinhas/sangue , Genoma/genética , Haplótipos/genética , Glicoproteínas de Membrana/genética , Família Multigênica/genética , Análise de Sequência de DNA , Homologia de Sequência , Sequências de Repetição em Tandem/genéticaRESUMO
Tapasin is an integral component of the peptide-loading complex (PLC) important for efficient peptide loading onto MHC class I molecules. We investigated the function of the tapasin-related protein, TAPBPR. Like tapasin, TAPBPR is widely expressed, IFN-γ-inducible, and binds to MHC class I coupled with ß2-microglobulin in the endoplasmic reticulum. In contrast to tapasin, TAPBPR does not bind ERp57 or calreticulin and is not an integral component of the PLC. ß2-microglobulin is essential for the association between TAPBPR and MHC class I. However, the association between TAPBPR and MHC class I occurs in the absence of a functional PLC, suggesting peptide is not required. Expression of TAPBPR decreases the rate of MHC class I maturation through the secretory pathway and prolongs the association of MHC class I on the PLC. The TAPBPR:MHC class I complex trafficks through the Golgi apparatus, demonstrating a function of TAPBPR beyond the endoplasmic reticulum/cis-Golgi. The identification of TAPBPR as an additional component of the MHC class I antigen-presentation pathway demonstrates that mechanisms controlling MHC class I expression remain incompletely understood.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Imunoglobulinas/metabolismo , Proteínas de Membrana/metabolismo , Apresentação de Antígeno/efeitos dos fármacos , Calnexina/metabolismo , Calreticulina/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Células HEK293 , Antígenos HLA-A/metabolismo , Células HeLa , Humanos , Interferon gama/farmacologia , Cinética , Proteínas de Membrana Transportadoras/metabolismo , Peptídeos/imunologia , Ligação Proteica/efeitos dos fármacos , Isomerases de Dissulfetos de Proteínas/metabolismo , Multimerização Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Microglobulina beta-2/metabolismoRESUMO
Contagious cancers that pass between individuals as an infectious cell line are highly unusual pathogens. Devil facial tumor disease (DFTD) is one such contagious cancer that emerged 16 y ago and is driving the Tasmanian devil to extinction. As both a pathogen and an allograft, DFTD cells should be rejected by the host-immune response, yet DFTD causes 100% mortality among infected devils with no apparent rejection of tumor cells. Why DFTD cells are not rejected has been a question of considerable confusion. Here, we show that DFTD cells do not express cell surface MHC molecules in vitro or in vivo, due to down-regulation of genes essential to the antigen-processing pathway, such as ß2-microglobulin and transporters associated with antigen processing. Loss of gene expression is not due to structural mutations, but to regulatory changes including epigenetic deacetylation of histones. Consequently, MHC class I molecules can be restored to the surface of DFTD cells in vitro by using recombinant devil IFN-γ, which is associated with up-regulation of the MHC class II transactivator, a key transcription factor with deacetylase activity. Further, expression of MHC class I molecules by DFTD cells can occur in vivo during lymphocyte infiltration. These results explain why T cells do not target DFTD cells. We propose that MHC-positive or epigenetically modified DFTD cells may provide a vaccine to DFTD. In addition, we suggest that down-regulation of MHC molecules using regulatory mechanisms allows evolvability of transmissible cancers and could affect the evolutionary trajectory of DFTD.
Assuntos
Espécies em Perigo de Extinção , Epigênese Genética/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade/imunologia , Marsupiais/imunologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/veterinária , Evasão Tumoral , Animais , Apresentação de Antígeno/imunologia , Linhagem Celular Tumoral , Interferon gama/imunologia , Neoplasias Cutâneas/patologiaRESUMO
The epithelial sodium channel (ENaC) of the kidney is necessary for extracellular volume homeostasis and normal arterial BP. Activity of ENaC is enhanced by proteolytic cleavage of the γ-subunit and putative release of a 43-amino acid inhibitory tract from the γ-subunit ectodomain. We hypothesized that proteolytic processing of γENaC occurs in the human kidney under physiologic conditions and that proteinuria contributes to aberrant proteolytic activation. Here, we used monoclonal antibodies (mAbs) with specificity to the human 43-mer inhibitory tract (N and C termini, mAbinhibit, and mAb4C11) and the neoepitope generated after proteolytic cleavage at the prostasin/kallikrein cleavage site (K181-V182 and mAbprostasin) to examine human nephrectomy specimens. By immunoblotting, kidney cortex homogenate from patients treated with angiotensin II type 1 receptor antagonists (n=6) or angiotensin-converting enzyme inhibitors (n=6) exhibited no significant difference in the amount of full-length or furin-cleaved γENaC or the furin-cleaved-to-full-length ratio of γENaC compared with homogenate from patients on no medication (n=5). Patients treated with diuretics (n=4) displayed higher abundance of full-length and furin-cleaved γENaC, with no significant change in the furin-cleaved-to-full-length γENaC ratio. In patients with proteinuria (n=6), the inhibitory tract was detected only in full-length γENaC by mAbinhibit. Prostasin/kallikrein-cleaved γENaC was detected consistently only in tissue from patients with proteinuria and observed in collecting ducts. In conclusion, human kidney γENaC is subject to proteolytic cleavage, yielding fragments compatible with furin cleavage, and proteinuria is associated with cleavage at the putative prostasin/kallikrein site and removal of the inhibitory tract within γENaC.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Rim/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Inibidores da Enzima Conversora de Angiotensina/química , Anticorpos/química , Anticorpos Monoclonais/química , Diuréticos/química , Exossomos/metabolismo , Feminino , Furina/química , Células HEK293 , Homeostase , Humanos , Calicreínas/química , Túbulos Renais Coletores/metabolismo , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Estrutura Terciária de Proteína , Sistema Renina-Angiotensina , Serina Endopeptidases/químicaRESUMO
The major histocompatibility complex (MHC) is a dynamic genomic region with an essential role in the adaptive immunity of jawed vertebrates. The evolution of the MHC has been dominated by gene duplication and gene loss, commonly known as the birth-and-death process. Evolutionary studies of the MHC have mostly focused on model species. However, the investigation of this region in non-avian reptiles is still in its infancy. To provide insights into the evolutionary mechanisms that have shaped the diversity of this region in the Order Crocodylia, we investigated MHC class I exon 3, intron 3, and exon 4 across 20 species of the families Alligatoridae and Crocodilidae. We generated 124 DNA sequences and identified 31 putative functional variants as well as 14 null variants. Phylogenetic analyses revealed three gene groups, all of which were present in Crocodilidae but only one in Alligatoridae. Within these groups, variants generally appear to cluster at the genus or family level rather than in species-specific groups. In addition, we found variation in gene copy number and some indication of interlocus recombination. These results suggest that MHC class I in Crocodylia underwent independent events of gene duplication, particularly in Crocodilidae. These findings enhance our understanding of MHC class I evolution and provide a preliminary framework for comparative studies of other non-avian reptiles as well as diversity assessment within Crocodylia.
Assuntos
Jacarés e Crocodilos/genética , Evolução Molecular , Genes MHC Classe I/genética , Variação Genética/genética , Jacarés e Crocodilos/classificação , Animais , Clonagem Molecular , DNA Complementar/genética , Filogenia , Reação em Cadeia da Polimerase , Recombinação GenéticaRESUMO
In most mammals, the MHC class I molecules are polymorphic and determine the specificity of peptide presentation, whereas the transporter associated with antigen presentation (TAP) heterodimers are functionally monomorphic. In chickens, there are two classical class I genes but only one is expressed at a high level, which can result in strong MHC associations with resistance to particular infectious pathogens. However, the basis for having a single dominantly expressed class I molecule has been unclear. Here we report TAP1 and TAP2 sequences from 16 chicken lines, and show that both genes have high allelic polymorphism and moderate sequence diversity, with variation in positions expected for peptide binding. We analyze peptide translocation in two MHC haplotypes, showing that chicken TAPs specify translocation at three peptide positions, matching the peptide motif of the single dominantly expressed class I molecule. These results show that coevolution between class I and TAP genes can explain the presence of a single dominantly expressed class I molecule in common chicken MHC haplotypes. Moreover, such coevolution in the primordial MHC may have been responsible for the appearance of the antigen presentation pathways at the birth of the adaptive immune system.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Galinhas/genética , Evolução Molecular , Antígenos de Histocompatibilidade Classe I/genética , Animais , Apresentação de Antígeno/genética , Dados de Sequência Molecular , Transporte ProteicoRESUMO
We have isolated a novel type of lectin named Arenicola marina lectin-1 (AML-1) from the lugworm A. marina. The lectin was purified from the coelomic fluid by affinity chromatography on a GlcNAc-derivatized column and eluted with GlcNAc. On SDS-PAGE, AML-1 showed an apparent molecular mass of 27 and 31 kDa in the reduced state. The N-terminal amino acid sequences were identical in these two bands. In the unreduced state, a complex band pattern was observed with bands from 35 kDa to more than 200 kDa. Two different full-length clones encoding polypeptides of 241 and 243 amino acids, respectively, were isolated from a coelomocyte cDNA library. The two clones, designated AML-1a and AML-1b, were 92% identical at the protein level and represent a novel type of protein sequence family. Purified AML-1 induced agglutination of rabbit erythrocytes, which could be inhibited by N-acetylated saccharides. Recombinant AML-1b showed the same band pattern as the native protein, whereas recombinant AML-1a in the reduced state lacked a 27 kDa band. AML-1b bound GlcNAc-derivatized columns and chitin, whereas AML-1a did not bind to these matrices. Immunohistochemical analysis revealed that AML-1 is expressed by coelomocytes in the nephridium and in round cells in the epidermis and in eggs. Moreover, AML-1 expression was up-regulated in response to a parasitic infection. We conclude that AML-1 purified from coelomic fluid is encoded by AML-1b and represents a novel type of protein family that binds acetylated components.