RESUMEN
The prostate is a glandular male accessory sex organ vital for normal fertility. It provides the prostatic component of seminal plasma which nourishes and protects sperm following ejaculation. Prostasomes are small (40-500 nm) membrane-bound vesicles produced by epithelial cells lining the prostate acini and are a component of prostatic secretions. Although the existence of these particles has been known for many years, their full function and relevance to reproductive health are largely unknown. Proteomic studies have shown a wide range of proteins (enzymes, structural proteins and novel, unannotated proteins) present in or on the surface of prostasomes providing them with a diverse nature. Interestingly prostasomes are able to fuse with sperm, this event and the associated transfer of proteins lies at the heart of many of their proposed functions. Sperm motility is increased by the presence of prostasomes and their fusion prevents premature acrosome reactions. Prostasomes have been shown to aid protection of sperm within the female reproductive tract because of immunosuppressive, antioxidant and antibacterial properties. Clinically these functions imply a role for prostasomes in male factor infertility. However, the very functions that promote fertility may have negative connotations in later life; recent work has suggested that prostasomes are involved in prostate cancer. Clearly more work is needed to clarify the role of these novel particles and their impact on men's health.
Asunto(s)
Vesículas Citoplasmáticas/fisiología , Vesículas Citoplasmáticas/ultraestructura , Fertilidad/fisiología , Infertilidad Masculina/fisiopatología , Próstata/fisiología , Reproducción/fisiología , Humanos , Masculino , Próstata/fisiopatología , Próstata/ultraestructura , Enfermedades de la Próstata/fisiopatologíaRESUMEN
BACKGROUND: Follicular aspirates represent a snapshot in time of conditions within the follicle at oocyte retrieval in women undergoing in vitro fertilization and embryo transfer. This clinical material has been much investigated and yet its cellular composition remains unclear. In this study we investigated the origin and profile of leukocytes found within follicular aspirates. METHODS: We performed morphological and immunohistochemical analyses of follicular aspirates and peripheral blood obtained concurrently at oocyte retrieval. RESULTS: There was no correlation between erythrocyte and leukocyte numbers in follicular aspirates. The profile of leukocyte subtypes within follicular aspirates was variable and differed significantly from the peripheral circulation in a significant proportion of the analysed samples. A subset of follicular aspirates displayed a marked increase in monocytes/macrophages and an apparent concomitant reduction in polymorphonuclear leukocytes compared with peripheral blood. CONCLUSIONS: Leukocytes within follicular aspirates cannot be accounted for solely as a result of blood vessel damage during oocyte retrieval. The variation in leukocyte subtypes observed in some follicular aspirates may reflect a coordinated infiltration of these cells, characteristic of progressive inflammatory responses in other systems. The possibility that leukocyte variation is indicative of follicular maturation deserves further investigation due to its potential relevance in optimizing oocyte selection.
Asunto(s)
Leucocitos/citología , Oocitos/citología , Folículo Ovárico/citología , Recolección de Tejidos y Órganos/métodos , Adulto , Transferencia de Embrión , Eritrocitos/citología , Femenino , Fertilización In Vitro/métodos , Humanos , Inmunohistoquímica , Leucocitos/metabolismo , Neutrófilos/citología , Oocitos/metabolismo , Folículo Ovárico/metabolismo , Factores de TiempoRESUMEN
The propensity of complement to damage self is controlled by expression of regulatory proteins. Recent results demonstrate that deleting just one of these regulators in mice causes complement to attack and destroy the embryo. These findings may have relevance to human pregnancy.
Asunto(s)
Proteínas del Sistema Complemento/inmunología , Embrión de Mamíferos/inmunología , Tolerancia Inmunológica , Placenta/inmunología , Receptores de Complemento/fisiología , Animales , Activación de Complemento , Femenino , Humanos , Ratones , Embarazo , Receptores de Complemento 3bRESUMEN
HLA-F is currently the most enigmatic of the human MHC-encoded class Ib genes. We have investigated the expression of HLA-F using a specific Ab raised against a synthetic peptide corresponding to amino acids 61-84 in the alpha1 domain of the predicted HLA-F protein. HLA-F is expressed as a beta2-microglobulin-associated, 42-kDa protein that shows a restricted tissue distribution. To date, we have detected this product only in peripheral blood B cells, B cell lines, and tissues containing B cells, in particular adult tonsil and fetal liver, a major site of B cell development. Thermostability assays suggest that HLA-F is expressed as an empty heterodimer devoid of peptide. Consistent with this, studies using endoglycosidase-H and cell surface immunoprecipitations also indicate that the overwhelming majority of HLA-F contains an immature oligosaccharide component and is expressed inside the cell. We have found that IFN-gamma treatment induces expression of HLA-F mRNA and HLA-F protein, but that this does not result in concomitant cell surface expression. HLA-F associates with at least two components of the conventional class I assembly pathway, calreticulin and TAP. The unusual characteristics of the predicted peptide-binding groove together with the predominantly intracellular localization raise the possibility that HLA-F may be capable of binding only a restricted set of peptides.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Antígenos HLA/biosíntesis , Antígenos HLA/aislamiento & purificación , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase I/aislamiento & purificación , Líquido Intracelular/inmunología , Líquido Intracelular/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Adulto , Secuencia de Aminoácidos , Presentación de Antígeno , Línea Celular , Regulación de la Expresión Génica/inmunología , Antígenos HLA/genética , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Interferón gamma/farmacología , Células Jurkat , Datos de Secuencia Molecular , Especificidad de Órganos/inmunología , Péptidos/inmunología , Péptidos/metabolismo , Unión Proteica/inmunología , Células Tumorales CultivadasRESUMEN
Human placental trophoblast is critically involved in mediating maternal tolerance of the fetal semiallograft. Genes encoding highly polymorphic major histocompatibility complex (MHC) class I and class II antigens that could provoke maternal immune rejection responses are silenced in trophoblast. However, several MHC class I or class I-related products exhibiting reduced or negligible polymorphism are expressed and assumed to be functionally involved in maintaining pregnancy. The CD1 gene family encodes non-polymorphic MHC class I-like products that have the unusual ability to present non-peptide antigens to T cells. One member, CD1D, is expressed in certain epithelial cells and interacts with a specific T-cell subset that may promote the development of Th2-mediated responses believed to be associated with pregnancy. In this study we examined the expression of CD1D in human trophoblast cell lines and placentally derived trophoblast cells by reverse transcriptase-polymerase chain reaction using CD1D-specific oligonucleotide primers. We have found that CD1D mRNA transcripts are expressed in trophoblast cells and cell lines. We have also identified a novel alternatively spliced CD1D mRNA transcript lacking exon 4. Exon 4-intact and exon 4-deficient CD1D transcripts appear to be differentially expressed in different trophoblast and non-trophoblast cell populations. Our studies suggest that at least one member of the CD1 family is transcribed in human trophoblast.
Asunto(s)
Antígenos CD1/genética , Coriocarcinoma/genética , Proteínas de Neoplasias/genética , Trofoblastos/metabolismo , Neoplasias Uterinas/genética , Antígenos CD1/metabolismo , Secuencia de Bases , Coriocarcinoma/metabolismo , Femenino , Humanos , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Embarazo , ARN Mensajero/genética , ARN Neoplásico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Células Tumorales Cultivadas , Neoplasias Uterinas/metabolismoRESUMEN
Neuroleptic Malignant Syndrome (NMS) is a life-threatening adverse reaction arising from the use of neuroleptic medications. While dopaminergic agonists, dantrolrene and supportive care are traditionally utilized in the stabilization and management of NMS, anticholinergic medication may also prove effective therapy. Treatment with anticholinergic medication has been suggested in cases of NMS associated with mild hyperthermia. We describe a case of 17-y-old female, who was brought to the emergency department for a possible "acute dystonic reaction". The patient received 50 mg diphenhydramine i.v., which resulted in improvement in mental status. The patient was readmitted to the emergency department 1 d following discharge with symptoms similar, but now considering the diagnosis of NMS. Diphenhydramine 50 mg i.v. was again administered and resulted in significant improvement.
Asunto(s)
Antagonistas Colinérgicos/uso terapéutico , Difenhidramina/uso terapéutico , Síndrome Neuroléptico Maligno/tratamiento farmacológico , Adolescente , Benzotropina/uso terapéutico , Femenino , Fiebre/complicaciones , Flufenazina/uso terapéutico , Humanos , Cloruro de Litio/uso terapéutico , Síndrome Neuroléptico Maligno/complicacionesRESUMEN
Human placental trophoblast expresses as unusual repertoire of major histocompatibility complex (MHC) class I products that appears to reflect the unique role of this epithelium in mediating feto-maternal relations during pregnancy. Trophoblast is devoid of human leucocyte antigen (HLA)-A,-B antigens but can express one or more non-HLA-A,-B class I proteins. The human choriocarcinoma cell lines JEG-3, BeWo and JAR are widely used as models to study trophoblast. During attempts to isolate non-HLA-A,-B class I from JEG-3 and BeWo by immunoaffinity chromatography using a monoclonal antibody to beta 2-microglobulin we observed a 55,000 MW protein co-purifying with class I. N-terminal amino acid sequencing and immunoblotting using a specific antiserum identified this product as calreticulin, a molecule recently shown to be involved in the assembly of classical class I in human B-lymphoblastoid cells. In our hands JEG-3 and BeWo were found to express 45,000 MW non-HLA-A,-B class I proteins while the 40,000 MW HLA-G product was identified only in JEG-3. Our data suggest that calreticulin associates with non-HLA-A,-B class I heterodimers and with free 45,000 MW non-HLA-A,-B class I H chains in JEG-3. JAR was found to be devoid of detectable class I H chains but contained beta 2-microglobulin and calreticulin. However, calreticulin-beta 2-microglobulin complexes were not detected in JAR. Calreticulin and class I were apparently co-localized within the endoplasmic reticulum of JEG-3 cells whereas only class I was expressed at the cell surface. These studies demonstrate that calreticulin is associated with non-HLA-A,-B class I products in human choriocarcinoma cells.
Asunto(s)
Autoantígenos , Proteínas de Unión al Calcio/análisis , Coriocarcinoma/metabolismo , Antígenos de Histocompatibilidad Clase I/análisis , Lectinas , Ribonucleoproteínas/análisis , Trofoblastos/inmunología , Neoplasias Uterinas/metabolismo , Calreticulina , Cromatografía de Afinidad , Femenino , Antígenos HLA/análisis , Antígenos HLA-G , Humanos , Immunoblotting , Microscopía Confocal , Células Tumorales CultivadasRESUMEN
The membrane-bound complement regulators decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46), and CD59 are broadly expressed proteins that act together to protect host tissues from autologous complement. Comparison of expression profiles of these proteins between normal and pathological tissues could reveal a mechanism by which tumor cells evade complement-mediated killing. Expression of the regulators was therefore examined in the normal human uterine cervix, in cervical intraepithelial neoplasia (CIN; n = 23), and in cervical squamous carcinomas (n = 6). DAF and MCP were reciprocally expressed in normal ectocervical epithelium. MCP was confined predominantly to the basal and parabasal layers with more extensive expression in metaplastic squamous epithelium. An apparent expansion in MCP expression was observed in more severe premalignant lesions whereas cervical carcinoma were uniformly MCP positive. By contrast, DAF expression appeared unaltered in premalignant lesions and variable in carcinomas. However, increased DAF was observed in stromal cells directly adjacent to infiltrating tumor cells. A low molecular weight DAF product was detected in tumors, and preliminary evidence suggests this may be derived from stromal cells. Overall, changes in expression of C3 convertase regulators in both the stromal and epithelial compartments may be important for evasion of immune surveillance in cervical cancer.
Asunto(s)
Antígenos CD/metabolismo , Antígenos CD55/metabolismo , Antígenos CD59/metabolismo , Carcinoma de Células Escamosas/metabolismo , Cuello del Útero/metabolismo , Glicoproteínas de Membrana/metabolismo , Lesiones Precancerosas/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Epitelio/metabolismo , Femenino , Humanos , Immunoblotting , Inmunohistoquímica , Masculino , Proteína Cofactora de Membrana , Embarazo , Valores de ReferenciaRESUMEN
The high-frequency blood group antigen Ok(a) is carried on a red cell membrane glycoprotein (gp) of 35-69 kDa that is widely distributed on malignant cells of different origins. Immunostaining of hemopoietic cells and a range of normal human tissues demonstrated a wide distribution of the Ok(a) gp that appears to be nonlineage-restricted, although certain tissues show differentiation-related expression. Ok(a) gp was purified from red cell membranes by immunoaffinity chromatography using mAb A103 and amino acid sequence analysis was performed. The N-terminal 30 amino acids are identical to the predicted sequence of M6 leukocyte activation antigen (M6), a member of the Ig superfamily (IgSF) with two IgSF domains. There are homologs in rat (MRC OX-47 or CE9), in mouse (basigin or gp42), and in chicken (HT7 or neurothelin). The molecular basis of the Ok(a) mutation was established by sequencing M6 cDNA derived from normal and Ok(a-) EBV-transformed B cell lines. A point mutation in the translated portion of M6 cDNA, G331AG-->AAG gives rise to a predicted E92-->K amino acid change in the first Ig-like domain of the Ok(a-) form of the protein. Transfection of mouse NS-0 cells with normal or Ok(a-) cDNA confirmed the identity of the protein and only the Ok(a-) transfectants failed to react with monoclonal anti-Ok(a) Ab.
Asunto(s)
Sistema del Grupo Sanguíneo ABO/sangre , Antígenos CD , Antígenos de Neoplasias , Antígenos de Superficie/sangre , Proteínas Aviares , Proteínas Sanguíneas , Inmunoglobulinas/química , Glicoproteínas de Membrana/sangre , Sistema del Grupo Sanguíneo ABO/biosíntesis , Sistema del Grupo Sanguíneo ABO/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Antígenos de Superficie/biosíntesis , Antígenos de Superficie/aislamiento & purificación , Basigina , Biomarcadores/sangre , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunoglobulinas/genética , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/aislamiento & purificación , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos/inmunología , RatasRESUMEN
The expression of HLA class I Ag by term human amnion epithelial cells was investigated. In immunostaining and FACS analysis, mAb to monomorphic class I Ag reacted extensively with amnion cells, whereas polymorphic mAb reactivity was more limited and variable. Further studies were conducted on amnion cell preparations containing negligible contaminants. Northern analysis with use of locus-specific probes demonstrated that amnion expresses two class Ib genes, HLA-E and HLA-G. Radio-immunoprecipitation with use of monomorphic mAb identified two fully glycosylated cell surface class I H chains of 44 and 41 kDa; polymorphic mAbs failed to immunoprecipitate the 41-kDa product, although 44-kDa products, typical of class Ia Ag, were identified in some preparations. Class I H chains were isolated from amnion by affinity chromatography. Microsequencing revealed that the first nine residues of the N-terminus of the 41-kDa product aligned perfectly only with HLA-E. Overall, amnion at term appears to express class Ib Ag with limited class Ia Ag. HLA-G is therefore expressed in two extrafetal epithelia: amnion and trophoblast. Identification of the class Ib protein HLA-E in amnion epithelium may have implications for preterm labor that can be associated with infection of the placental membranes.
Asunto(s)
Amnios/inmunología , Antígenos HLA/biosíntesis , Antígenos HLA/genética , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase I/genética , Amnios/citología , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Northern Blotting , Células Cultivadas , Cromatografía de Afinidad , Femenino , Citometría de Flujo , Regulación de la Expresión Génica/inmunología , Glicósido Hidrolasas/fisiología , Antígenos HLA/inmunología , Antígenos HLA-G , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Immunoblotting , Datos de Secuencia Molecular , Pruebas de Precipitina , Trofoblastos/citología , Antígenos HLA-ERESUMEN
Glycoproteins expressing the Lutheran blood group antigens were isolated from human erythrocyte membranes and from human fetal liver. Amino acid sequence analyses allowed the design of redundant oligonucleotides that were used to generate a 459-bp, sequence-specific probe by PCR. A cDNA clone of 2400 bp was isolated from a human placental lambda gt 11 library and sequenced, and the deduced amino acid sequence was studied. The predicted mature protein is a type I membrane protein of 597 amino acids with five potential N-glycosylation sites. There are five disulfide-bonded, extracellular, immunoglobulin superfamily domains (two variable-region set and three constant-region set), a single hydrophobic, membrane-spanning domain, and a cytoplasmic domain of 59 residues. The overall structure is similar to that of the human tumor marker MUC 18 and the chicken neural adhesion molecule SC1. The extracellular domains and cytoplasmic domain contain consensus motifs for the binding of integrin and Src homology 3 domains, respectively, suggesting possible receptor and signal-transduction function. Immunostaining of human tissues demonstrated a wide distribution and provided evidence that the glycoprotein is under developmental control in liver and may also be regulated during differentiation in other tissues.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Hígado/metabolismo , Sistema del Grupo Sanguíneo Lutheran/genética , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Secuencia de Bases , Membrana Celular/metabolismo , Mapeo Cromosómico , Cromosomas Humanos Par 19 , ADN Complementario , Eritrocitos/metabolismo , Genes de Inmunoglobulinas , Humanos , Inmunohistoquímica , Hígado/embriología , Sistema del Grupo Sanguíneo Lutheran/química , Datos de Secuencia Molecular , Trofoblastos/metabolismoRESUMEN
Three synthetic peptides encompassing the entire cytoplasmic polypeptide sequence (amino acid residues 82-128) of glycophorin C (GPC) and glycophorin D (GPD) were used to immunize mice for the production of monoclonal antibodies (MoAbs). Only the synthetic peptide (GPC-peptide-1) corresponding to C-terminal residues 112-128 elicited a MoAb (named BGRL-100) which could react with native and denatured GPC and GPD. We characterized BGRL-100 by inhibition using GPC-peptide 1 and red cell sialoglycoproteins. The ability of BGRL-100 to interact with native GPC and GPD was assessed by immunoprecipitation with normal red cells (RBCs), and with denatured GPC and GPD by Western blotting of both normal RBCs and RBCs carrying GPC variants. Immunohistochemical staining of human tissue sections was performed using both BGRL-100 and a rat MoAb (named BRAC-1), which is specific for an extracellular domain of GPC and GPD. Both antibodies showed strong staining of erythroid lineage haemopoietic cells in fetal liver, sinusoids of adult liver and RBCs in the blood vessels of all tissues tested. Neither antibody reacted with epithelia from a range of human tissues. However, both MoAbs stained neural tissue in a distinctive fibrillar pattern. This suggests the presence of an analogue of erythroid GPC in neural tissues.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Eritrocitos/inmunología , Glicoforinas/inmunología , Fragmentos de Péptidos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Unión Competitiva , Western Blotting , Glicoforinas/análisis , Células Madre Hematopoyéticas/química , Humanos , Hígado/química , Hígado/embriología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Pruebas de Precipitina , RatasRESUMEN
The CD47 glycoprotein was isolated from human erythrocytes by immunoprecipitation using monoclonal antibody (mAb) BRIC-125. Enzymic deglycosylation of the protein showed it contained N-linked oligosaccharides, and trypsin proteolysis of the protein in situ in the erythrocyte membrane cleaved it into two portions, one of which was glycosylated. Both the intact protein and the glycosylated fragment had blocked N-termini. Amino acid sequence was obtained from several proteolytic fragments of CD47. Comparison with the sequence database showed the protein to be very similar to or identical with OA3, a multispanning membrane protein. The protein also appears to be the same as the integrin-associated protein, which has a role in cell adhesion in non-erythroid cells. CD47 has six potential N-glycosylation sites, five of which are in an Ig superfamily domain. We show that three of these sites carry N-glycans in erythrocytes. Immunocytochemical staining of human tissues showed that CD47 was broadly distributed on mesenchyme and epithelia at multiple sites. Reactivity was particularly prominent in surface and ductular epithelia, and in the brain. The possible roles of the CD47 glycoprotein are discussed.
Asunto(s)
Antígenos CD/sangre , Proteínas Portadoras/sangre , Eritrocitos/química , Proteínas de la Membrana/sangre , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Antígenos CD/química , Química Encefálica , Antígeno CD47 , Conformación de Carbohidratos , Proteínas Portadoras/química , Femenino , Glicosilación , Humanos , Técnicas de Inmunoadsorción , Hígado/química , Datos de Secuencia Molecular , Oligosacáridos/análisis , Neoplasias Ováricas/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Homología de Secuencia , Distribución Tisular , Tripsina/metabolismoRESUMEN
The seminal plasma complement regulator membrane cofactor protein (MCP) was examined by sequential centrifugation and phase separation in the detergent Triton X-114. The presence of MCP components in seminal plasma depleted of the 40 kDa sperm MCP product by low speed centrifugation was confirmed. Subsequent centrifugation at 2200 g recovered a pellet containing a prominent 60 kDa and a weak 50-55 kDa MCP component. A 60 kDa MCP product remained detectable in the supernatant fraction after this centrifugation step but this was depleted by ultracentrifugation. Recovery of the seminal plasma MCP components in the pellet fraction obtained by ultracentrifugation suggested that seminal plasma MCP is membrane-associated. Seminal plasma fractions were also subjected to phase separation in Triton X-114. MCP components in both pellet and supernatant fractions partitioned to the detergent phase, confirming that seminal plasma MCP is membrane associated. The origin of these proteins was investigated by analysing MCP products in seminal plasma from vasectomized men. The 40 kDa sperm MCP protein was absent but a 60 kDa MCP component, which partitioned to the detergent phase in Triton X-114, was evident. Seminal plasma therefore contains typical membrane-associated MCP products that appear to be derived distal to the ductus deferens.
Asunto(s)
Antígenos CD/análisis , Proteínas del Sistema Complemento/inmunología , Glicoproteínas de Membrana/análisis , Semen/inmunología , Membrana Celular/inmunología , Centrifugación , Detergentes , Humanos , Immunoblotting , Masculino , Proteína Cofactora de Membrana , Métodos , Octoxinol , Polietilenglicoles , VasectomíaRESUMEN
We have examined the distribution of the complement (C) regulatory proteins CD59, membrane cofactor protein (MCP) and decay-accelerating factor (DAF) on mature sperm and compared expression of these proteins in parallel both during spermatogenesis and in the prostate. Enhanced immunoperoxidase staining and radioimmunoassay confirmed that C regulators are differentially expressed on sperm; CD59 was strongly expressed on the surface of acrosome intact sperm while MCP and DAF appear to be located primarily on the inner acrosomal membrane. While the MW of CD59 on sperm is typical of other systems, we confirm that in addition to a novel 40,000-46,000 MW MCP protein, sperm also express a novel 55,000 MW DAF product. Examination of normal testis by immunostaining revealed that although C regulators are differentially expressed within the germinal epithelium, all three proteins were present on the acrosomal region of condensing spermatids. We show that novel, low MW forms of MCP and DAF are expressed in normal testis membranes but are absent from testis membranes obtained from patients undergoing gender reassignment surgery in whom the germinal epithelium is diminished. Novel MW C3 convertase regulators are therefore associated with differentiating germinal epithelium. Typical CD59 components were also present on normal testis membranes confirming that CD59 is acquired during spermatogenesis. We demonstrate that the prostatic epithelium, in addition to MCP, expresses CD59 but not DAF. By comparison with CD59, therefore, our studies suggest that DAF may be acquired only in the testis. Overall, our data suggest that, on leaving the testis, sperm express the repertoire of C regulators required for protection from C during their transit through the male and female reproductive tracts.
Asunto(s)
Antígenos CD/análisis , Proteínas Inactivadoras de Complemento/análisis , Glicoproteínas de Membrana/análisis , Espermatogénesis/inmunología , Acrosoma/inmunología , Western Blotting , Antígenos CD55 , Antígenos CD59 , Humanos , Técnicas para Inmunoenzimas , Masculino , Proteína Cofactora de Membrana , Próstata/inmunología , Espermatozoides/inmunología , Testículo/inmunologíaRESUMEN
Expression of Fc gamma receptors on human placental trophoblast was investigated by immunostaining and immunoblotting using a panel of Fc gamma receptor monoclonal antibodies (mAb). Fc gamma receptors typical of other cell types were not detected on syncytiotrophoblast in term placentae when transplacental IgG transport was maximal. Unexpectedly, however, and by contrast with term, all Fc gamma receptor III mAb tested bound to first trimester placental syncytiotrophoblast by immunostaining. Reactivity was relatively restricted and varied between specimens. Fc gamma receptor III products of 41,000-45,000 and 49,000-52,000 MW were consistently detected on first trimester trophoblast membranes by immunoblotting and levels of these products were greatly reduced following treatment with phosphatidylinositol-specific phospholipase C, suggesting that the early trophoblast Fc gamma receptor III is glycosyl-phosphatidylinositol (GPI) linked. The mAb Leu-11b behaved differently to other anti-Fc gamma receptor III mAb examined. By immunostaining, Leu-11b bound to syncytiotrophoblast at term and detected both syncytiotrophoblast and underlying cytotrophoblast in the first trimester. In addition to the GPI-anchored Fc gamma receptor III in first trimester, Leu-11b also detected a 74,000 MW component on both first trimester and term trophoblast membranes by immunoblotting. Thus trophoblast appears to express a GPI-anchored Fc gamma receptor III in first trimester but not term placentae. With the exception of the 74,000 MW Leu-11b-defined product whose function is unclear, currently available Fc gamma receptor mAb appear to be incapable of detecting the protein involved in IgG transport during the later stages of gestation.
Asunto(s)
Receptores de IgG/análisis , Trofoblastos/inmunología , Anticuerpos Monoclonales/inmunología , Vellosidades Coriónicas/inmunología , Femenino , Humanos , Immunoblotting , Técnicas para Inmunoenzimas , Peso Molecular , Embarazo , Primer Trimestre del Embarazo , Tercer Trimestre del Embarazo , Receptores de IgG/químicaRESUMEN
The human fetus appears to be capable of protecting itself from maternal complement (C) from an early stage in development by expressing the C regulatory proteins decay-accelerating factor (DAF), membrane co-factor protein (MCP) and CD59 on fetally derived trophoblast at the feto-maternal interface. In this study we have examined the ontogeny of these proteins within the fetus itself and have focused on the liver which represents a major site of haemopoiesis during development. Immunostaining revealed that DAF, MCP and CD59 are all expressed from at least 6 weeks of gestation in the liver but that these proteins display distinct distribution patterns. CD59 was broadly distributed both within the epithelial and haemopoietic compartments, but expression of C3 convertase regulators was more restricted. DAF expression was limited to isolated cells within haemopoietic nests and the epithelium was DAF-negative. Although MCP expression on haemopoietic cells was also limited, by contrast with DAF the developing hepatic epithelium was strongly MCP-positive. Typical CD59 and MCP components were observed in fetal liver extracts by immunoblotting, although liver MCP components consistently migrated 4000-5000 MW ahead of those observed on placental trophoblast. Differences in the distribution of these proteins were also observed between the fetal and adult liver. In particular, by comparison with fetal hepatic epithelium, there was an apparent loss of MCP expression from adult hepatocytes. Thus, MCP appears to be developmentally regulated in the human liver and is expressed in the absence of DAF on the early hepatic epithelium. Overall, this study suggests that C regulatory proteins, and in particular CD59 and MCP, are required from the very early stages of gestation within the fetus itself.
Asunto(s)
Antígenos CD/análisis , Proteínas Inactivadoras de Complemento/análisis , Hígado/embriología , Glicoproteínas de Membrana/análisis , Adulto , Envejecimiento/inmunología , Western Blotting , Antígenos CD55 , Antígenos CD59 , Proteínas del Sistema Complemento/inmunología , Humanos , Técnicas para Inmunoenzimas , Hígado/inmunología , Proteína Cofactora de MembranaRESUMEN
Recent studies have revealed that human trophoblast expresses three membrane-bound proteins which function specifically to regulate the activity of complement. These proteins are already known to be widely distributed in normal adult tissues where they protect host cells from damage resulting from the fortuitous deposition of activated complement components. Their activities are focused at two distinct steps in the complement pathway. Decay accelerating factor (DAF, CD55) and membrane co-factor protein (MCP, CD46) act at the level of the C3 convertase enzymes which activate C3 to C3b. A further protein, CD59, directly regulates the formation and function of the terminal cytolytic membrane attack complex (MAC) by specifically interacting with C8 and C9. These proteins appear to play an important role in the maintenance of normal human pregnancy. DAF, MCP and CD59 are all expressed where trophoblast surfaces are in contact with maternal blood and tissues and expression occurs from at least 6 weeks of gestation. The semi-allogeneic human conceptus therefore appears to be effectively protected from maternal complement-mediated damage arising either from alternative or classical pathway activation or in a bystander fashion following a response to microbial infection in the mother. Complement regulatory protein deficiency disorders with clinically demonstrable consequences especially in terms of haemolytic disease are known to exist and have proved valuable in establishing the biological role of these proteins in vivo. The demonstration of this new family of immunoregulatory proteins on trophoblast raises important questions about the potential involvement of these products in pregnancy pathologies.
Asunto(s)
Proteínas del Sistema Complemento/inmunología , Feto/inmunología , Embarazo/inmunología , Complemento C3/inmunología , Convertasas de Complemento C3-C5/inmunología , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Vía Alternativa del Complemento , Vía Clásica del Complemento , Femenino , Humanos , Complicaciones del Embarazo/inmunología , Trofoblastos/inmunologíaRESUMEN
HLA class I Ag expression was investigated in the human fetal liver. By immunostaining, mAb to monomorphic class I Ag showed widespread reactivity with both epithelial and hemopoietic cells. By contrast, mAb to polymorphic determinants showed more restricted reactivity that was confined to a proportion of hemopoietic cells: the hepatic epithelium was essentially unreactive. This suggested that the developing liver might express nonclassical HLA class I. Class I Ag were examined in membrane and cytosol fractions of mid-trimester fetal liver. Because of its broad reactivity with HLA-A,-B, mAb Q1/28 was selected to identify classical class I Ag in these studies. Immunoprecipitations were carried out against radiolabeled glycoprotein extracts of fetal liver membranes. W6/32 detected a 40-kDa product characteristic of nonclassical class I proteins, as well as a 43-kDa product, in lysates immunodepleted with Q1/28. By immunoblotting, an anti-H chain antiserum (HC) identified a Q1/28- 40-kDa component and a 43-kDa Q1/28+ component in fetal liver membrane glycoproteins. The fetal liver cytosol fraction was found to contain a 42- to 43-kDa product by W6/32-chromatography. This component partitioned to the aqueous phase upon condensation in TritonX-114 detergent and by immunoblotting was reactive with monomorphic mAb HC10 but not with Q1/28. Total RNA and polymerase chain reaction amplified class I transcripts of fetal liver were probed using oligonucleotides specific for HLA-E, -F, and -G. HLA-F, was readily detected in total RNAs by Northern analysis. HLA-E, HLA-F, and HLA-G were all detected in fetal liver by polymerase chain reaction. Differential expression of these genes may occur between the first and second trimester of liver development. Overall therefore, the human fetal liver expresses multiple class I protein products and contains transcripts for non-classical class I genes; particularly HLA-F.
Asunto(s)
Feto/inmunología , Expresión Génica , Genes MHC Clase I , Hígado/inmunología , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Femenino , Antígenos de Histocompatibilidad Clase I/análisis , Antígenos de Histocompatibilidad Clase I/fisiología , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , EmbarazoRESUMEN
The complement (C) regulatory proteins decay-accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), which control C3 convertases, together with CD59, an inhibitor of the membrane attack complex (MAC), were found to be present in the developing human placenta from at least 6 weeks of gestation until term. Immunostaining revealed differences in the distribution of these proteins on the fetally derived trophoblast epithelium, especially in early placentae which contain trophoblast populations of diverse proliferative potential and differentiation status. Expression of all three proteins occurred on the terminally differentiated syncytiotrophoblast epithelium covering chorionic villi and which is in direct contact with maternal blood. CD59 was also expressed on the underlying villous cytotrophoblast cells and on their extra-villous derivatives. These two populations showed differential expression of the C3 convertase regulators. Villous cytotrophoblast cells expressed MCP but were largely devoid of DAF. Proliferation of this population to generate extra-villous cytotrophoblast cell columns was associated with both an increase in DAF expression and a decrease in MCP expression. Throughout placental development, expression of DAF appeared to be lower than that of MCP and CD59 as assessed by solid-phase binding assays on isolated trophoblast membranes. Early placentae were also found to contain both DAF+ and DAF- chorionic villi. Conversely, expression of CD59 appeared comparatively high and transcripts for CD59 were found to be much more abundant than those for DAF in purified trophoblast cells. C regulatory proteins appear to play an important role throughout gestation in protecting the fetally derived human conceptus from maternal C. The differential expression patterns of the proteins on trophoblast may reflect differences in requirement for specific functional activities at different locations within the placenta.