RESUMO
Traumatic brain injury (TBI) leads to lasting brain dysfunction with chronic neuroinflammation typified by nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 (NLRP3) inflammasome activation in microglia. This study probed whether a single intranasal (IN) administration of human mesenchymal stem cell-derived extracellular vesicles (hMSC-EVs) naturally enriched with activated microglia-modulating miRNAs can avert chronic adverse outcomes of TBI. Small RNA sequencing confirmed the enrichment of miRNAs capable of modulating activated microglia in hMSC-EV cargo. IN administration of hMSC-EVs into adult mice ninety minutes after the induction of a unilateral controlled cortical impact injury resulted in their incorporation into neurons and microglia in both injured and contralateral hemispheres. A single higher dose hMSC-EV treatment also inhibited NLRP3 inflammasome activation after TBI, evidenced by reduced NLRP3, apoptosis-associated speck-like protein containing a CARD, activated caspase-1, interleukin-1 beta, and IL-18 levels in the injured brain. Such inhibition in the acute phase of TBI endured in the chronic phase, which could also be gleaned from diminished NLRP3 inflammasome activation in microglia of TBI mice receiving hMSC-EVs. Proteomic analysis and validation revealed that higher dose hMSC-EV treatment thwarted the chronic activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway by IL-18, which decreased the release of proinflammatory cytokines. Inhibition of the chronic activation of NLRP3-p38/MAPK signaling after TBI also prevented long-term cognitive and mood impairments. Notably, the animals receiving higher doses of hMSC-EVs after TBI displayed better cognitive and mood function in all behavioral tests than animals receiving the vehicle after TBI. A lower dose of hMSC-EV treatment also partially improved cognitive and mood function. Thus, an optimal IN dose of hMSC-EVs naturally enriched with activated microglia-modulating miRNAs can inhibit the chronic activation of NLRP3-p38/MAPK signaling after TBI and prevent lasting brain dysfunction.
Assuntos
Lesões Encefálicas Traumáticas , Vesículas Extracelulares , MicroRNAs , Proteína Quinase 14 Ativada por Mitógeno , Animais , Humanos , Camundongos , Encéfalo/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Vesículas Extracelulares/metabolismo , Inflamassomos/metabolismo , Interleucina-18/metabolismo , MicroRNAs/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteômica , Transdução de Sinais , Células-Tronco MesenquimaisRESUMO
Neurotoxic side effects of chemotherapy include deficits in attention, memory, and executive functioning. Currently, there are no FDA-approved therapies. In mice, cisplatin causes long-term cognitive deficits, white matter damage, mitochondrial dysfunction, and loss of synaptic integrity. We hypothesized that MSC-derived small extracellular vesicles (sEVs) could restore cisplatin-induced cognitive impairments and brain damage. Animals were injected with cisplatin intraperitoneally and treated with MSC-derived sEVs intranasally 48 and 96 h after the last cisplatin injection. The puzzle box test (PBT) and the novel object place recognition test (NOPRT) were used to determine cognitive deficits. Synaptosomal mitochondrial morphology was analyzed by transmission electron microscopy. Immunohistochemistry using antibodies against synaptophysin and PSD95 was applied to assess synaptic loss. Black-Gold II staining was used to quantify white matter integrity. Our data show that sEVs enter the brain in 30 min and reverse the cisplatin-induced deficits in executive functioning and working and spatial memory. Abnormalities in mitochondrial morphology, loss of white matter, and synaptic integrity in the hippocampus were restored as well. Transcriptomic analysis revealed upregulation of regenerative functions after treatment with sEVs, pointing to a possible role of axonal guidance signaling, netrin signaling, and Wnt/Ca2+ signaling in recovery. Our data suggest that intranasal sEV treatment could become a novel therapeutic approach for the treatment of chemobrain.
Assuntos
Transtornos Cognitivos , Disfunção Cognitiva , Vesículas Extracelulares , Camundongos , Animais , Cisplatino/efeitos adversos , Disfunção Cognitiva/induzido quimicamente , Disfunção Cognitiva/terapia , Encéfalo , Transtornos Cognitivos/induzido quimicamenteRESUMO
Reports of neurological sequelae related to colon cancer are largely restricted to rare instances of paraneoplastic syndromes, due to autoimmune reactions. Systemic inflammation associated with tumor development influences sensory neuron function in other disease models, though the extent to which this occurs in colorectal cancer is unknown. We induced orthotopic colorectal cancer via orthotopic injection of two colorectal cancer cell lines (MC38 and CT26) in two different mouse strains (C57BL/6 and Balb/c, respectively). Behavioral tests of pain sensitivity and activity did not detect significant alterations in sensory sensitivity or diminished well-being throughout tumor development. However, immunohistochemistry revealed widespread reductions in intraepidermal nerve fiber density in the skin of tumor-bearing mice. Though loss of nerve fiber density was not associated with increased expression of cell injury markers in dorsal root ganglia, lumbar dorsal root ganglia neurons of tumor-bearing animals showed deficits in mitochondrial function. These neurons also had reduced cytosolic calcium levels in live-cell imaging and reduced spontaneous activity in multi-electrode array analysis. Bulk RNA sequencing of DRGs from tumor-bearing mice detected activation of gene expression pathways associated with elevated cytokine and chemokine signaling, including CXCL10. This is consistent with the detection of CXCL10 (and numerous other cytokines, chemokines and growth factors) in MC38 and CT26 cell-conditioned media, and the serum of tumor-bearing mice. Our study demonstrates in a pre-clinical setting that colon cancer is associated with latent sensory neuron dysfunction and implicates cytokine/chemokine signaling in this process. These findings may have implications for determining risk factors and treatment responsiveness related to neuropathy in colorectal cancer.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Animais , Neoplasias do Colo/complicações , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Neoplasias Colorretais/complicações , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Gânglios Espinais/metabolismo , Hiperalgesia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células Receptoras Sensoriais/metabolismoRESUMO
Acute kidney injury (AKI) is a sudden decline of renal function and represents a global clinical problem due to an elevated morbidity and mortality. Despite many efforts, currently there are no treatments to halt this devastating condition. Extracellular vesicles (EVs) are nanoparticles secreted by various cell types in both physiological and pathological conditions. EVs can arise from distinct parts of the kidney and can mediate intercellular communication between various cell types along the nephron. Besides their potential as diagnostic tools, EVs have been proposed as powerful new tools for regenerative medicine and have been broadly studied as therapeutic mediators in different models of experimental AKI. In this review, we present an overview of the basic features and biological relevance of EVs, with an emphasis on their functional role in cell-to-cell communication in the kidney. We explore versatile roles of EVs in crucial pathophysiological mechanisms contributing to AKI and give a detailed description of the renoprotective effects of EVs from different origins in AKI. Finally, we explain known mechanisms of action of EVs in AKI and provide an outlook on the potential clinical translation of EVs in the setting of AKI.
Assuntos
Injúria Renal Aguda , Vesículas Extracelulares , Células-Tronco Mesenquimais , Injúria Renal Aguda/patologia , Vesículas Extracelulares/metabolismo , Humanos , Rim/metabolismo , Células-Tronco Mesenquimais/metabolismoRESUMO
Mucin 16 (MUC16) is a transmembrane type mucin and its released extracellular portion is designated as CA125 antigen. It is considered to be part of a supramolecular glycoprotein complex having a complicated epitope map and extreme structural heterogeneity. Starting from the initial transmembrane localization of MUC16/CA125 antigen and its alternative routes of release by shedding or putative secretion, CA125 antigen from human amniotic fluid soluble and extracellular vesicles (EVs)-containing fractions were characterized aiming at the possible glycosylation-associated mode of distribution as a factor contributing to the reported conflicting structural data. Ultracentrifugation, sucrose density gradient centrifugation, ion-exchange chromatography and TEM were used for analysis. The results indicated that the smeared abundantly glycosylated high molecular mass CA125-immunoreactive species, which follow the wheat germ agglutinin-binding pattern, were shared across amniotic fluid soluble and particulate fractions. A lower molecular mass glycoprotein-like CA125-immunoreactive species which follows the peanut agglutinin-binding pattern and was specifically associated with the EVs-enriched fraction was observed. CA125 presentation in the particulate amniotic fluid fraction was found to be shaped by a complex interactome partially involving lactose-sensitive galectin-3 binding. The MUC16 - EVs alliance as well as heterogeneous mucin/macromolecular complexes, at membranes or extracellularly, may represent cryptic pools of distinct CA125 species.
Assuntos
Antígeno Ca-125/metabolismo , Vesículas Extracelulares/metabolismo , Mucina-1/metabolismo , Líquido Amniótico/metabolismo , Centrifugação com Gradiente de Concentração , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Galectina 3/metabolismo , Glicosilação , Humanos , Microscopia Eletrônica de Transmissão , Ácidos Nucleicos Peptídicos/metabolismo , Ultracentrifugação , Aglutininas do Germe de Trigo/metabolismoRESUMO
In the hypothalamus, insulin takes on many roles involved in energy homoeostasis. Therefore, the aim of this study was to examine hypothalamic insulin expression during the initial phase of the metabolic response to fasting. Hypothalamic insulin content was assessed by both radioimmunoassay and Western blot. The relative expression of insulin mRNA was examined by qPCR. Immunofluorescence and immunohistochemistry were used to determine the distribution of insulin immunopositivity in the hypothalamus. After 6-h fasting, both glucose and insulin levels were decreased in serum but not in the cerebrospinal fluid. Our study showed for the first time that, while the concentration of circulating glucose and insulin decreased, both insulin mRNA expression and insulin content in the hypothalamic parenchyma were increased after short-term fasting. Increased insulin immunopositivity was detected specifically in the neurons of the hypothalamic periventricular nucleus and in the ependymal cells of fasting animals. These novel findings point to the complexity of mechanisms regulating insulin expression in the CNS in general and in the hypothalamus in particular.
Assuntos
Jejum/metabolismo , Hipotálamo/metabolismo , Insulina/metabolismo , Animais , Glicemia/metabolismo , Jejum/sangue , Jejum/líquido cefalorraquidiano , Insulina/sangue , Insulina/líquido cefalorraquidiano , Insulina/genética , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
This study was aimed at evaluation of the contribution of acid-soluble glycoproteins (ASG)/mucins and extracellular vesicles (EVs), yet unexplored components of human seminal plasma (hSP) to the complexity of its glycome. Gaining insight into the native presentation and distribution of glycans across hSP could help establish molecular environments supporting specific biological activities based on unique ligand capacities. Soluble and particulate fractions of hSP from healthy subjects were analyzed by gel filtration, electrophoresis, ion-exchange chromatography and a solid phase assay with immobilized charge-resolved glycospecies to test their reactivity with plant lectins, carbohydrate-binding antibodies and selected human lectins. Common O- and N-glycosylated species were detected on mixed or overlapped underlying protein scaffolds in both soluble and particulate fractions of hSP. Siaα2,6Gal and N-glycans were concentrated on EVs, whereas Siaα2,3Gal, T and Tn antigens were selectively associated with distinct glycospecies of ASG/mucins. Accessible ligands for the lectins, DC-SIGN and Siglec-9, were detected in all hSP components, but they preferentially bound to EVs glycospecies. Insight into the complexity of hSP glycans as recognition signals under normal physiological conditions could be of interest for regulation and possible modulation of its biological activity, as well as for biomarker potential related to male health.
Assuntos
Glicoproteínas/química , Mucinas/química , Sêmen/química , Antígenos CD/química , Antígenos Glicosídicos Associados a Tumores/química , Biomarcadores/química , Biotinilação , Moléculas de Adesão Celular/química , Cromatografia em Gel , Glicosilação , Humanos , Lectinas Tipo C/química , Ligantes , Masculino , Percloratos/química , Polissacarídeos/química , Receptores de Superfície Celular/química , Proteínas Recombinantes/química , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/químicaRESUMO
CA-125 (coelomic epithelium-related antigen) forms the extracellular portion of transmembrane mucin 16 (MUC16). It is shed after proteolytic degradation. Due to structural heterogeneity, CA-125 ligand capacity and biological roles are not yet understood. In this study, we assessed CA-125 as a ligand for dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN), which is a C-type lectin showing specificity for mannosylated and fucosylated structures. It plays a role as a pattern recognition molecule for viral and bacterial glycans or as an adhesion receptor. We probed a human DC-SIGN-Fc chimera with CA-125 of fetal or cancer origin using solid- or fluid-phase binding and inhibition assays. The results showed that DC-SIGN binds to CA-125 of fetal origin and that this interaction is carbohydrate-dependent. By contrast, cancer-derived CA-125 displayed negligible binding. Inhibition assays indicated differences in the potency of CA-125 to interfere with DC-SIGN binding to pathogen-related glycoconjugates, such as mannan and Helicobacter pylori antigens. The differences in ligand properties between CA-125 of fetal and cancer origin may be due to specificities of glycosylation. This might influence various functions of dendritic cells based on their subset diversity and maturation-related functional capacity.
Assuntos
Antígeno Ca-125/metabolismo , Moléculas de Adesão Celular/metabolismo , Feto/metabolismo , Lectinas Tipo C/metabolismo , Receptores de Superfície Celular/metabolismo , Antígeno Ca-125/química , Antígeno Ca-125/isolamento & purificação , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Glicoconjugados/química , Glicoconjugados/metabolismo , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Lectinas Tipo C/química , Lectinas Tipo C/genética , Ligantes , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
Kidney disease is a growing public health problem worldwide, including both acute and chronic forms. Existing therapies for kidney disease target various pathogenic mechanisms; however, these therapies only slow down the progression of the disease rather than offering a cure. One of the potential and emerging approaches for the treatment of kidney disease is mesenchymal stromal/stem cell (MSC) therapy, shown to have beneficial effects in preclinical studies. In addition, extracellular vesicles (EVs) released by MSCs became a potent cell-free therapy option in various preclinical models of kidney disease due to their regenerative, anti-inflammatory, and immunomodulatory properties. However, there are scarce clinical data available regarding the use of MSC-EVs in kidney pathologies. This review article provides an outline of the renoprotective effects of MSC-EVs in different preclinical models of kidney disease. It offers a comprehensive analysis of possible mechanisms of action of MSC-EVs with an emphasis on kidney disease. Finally, on the journey toward the implementation of MSC-EVs into clinical practice, we highlight the need to establish standardized methods for the characterization of an EV-based product and investigate the adequate dosing, safety, and efficacy of MSC-EVs application, as well as the development of suitable potency assays.
RESUMO
An optimal intranasal (IN) dose of human mesenchymal stem cell-derived extracellular vesicles (hMSC-EVs), 90 min post-traumatic brain injury (TBI), has been reported to prevent the evolution of acute neuroinflammation into chronic neuroinflammation resulting in the alleviation of long-term cognitive and mood impairments. Since hippocampal neurogenesis decline and synapse loss contribute to TBI-induced long-term cognitive and mood dysfunction, this study investigated whether hMSC-EV treatment after TBI can prevent hippocampal neurogenesis decline and synapse loss in the chronic phase of TBI. C57BL6 mice undergoing unilateral controlled cortical impact injury (CCI) received a single IN administration of different doses of EVs or the vehicle at 90 min post-TBI. Quantifying neurogenesis in the subgranular zone-granule cell layer (SGZ-GCL) through 5'-bromodeoxyuridine and neuron-specific nuclear antigen double labeling at ~2 months post-TBI revealed decreased neurogenesis in TBI mice receiving vehicle. However, in TBI mice receiving EVs (12.8 and 25.6 × 109 EVs), the extent of neurogenesis was matched to naive control levels. A similar trend of decreased neurogenesis was seen when doublecortin-positive newly generated neurons were quantified in the SGZ-GCL at ~3 months post-TBI. The above doses of EVs treatment after TBI also reduced the loss of pre-and post-synaptic marker proteins in the hippocampus and the somatosensory cortex. Moreover, at 48 h post-treatment, brain-derived neurotrophic factor (BDNF), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), and phosphorylated cyclic AMP response-element binding protein (p-CREB) levels were downregulated in TBI mice receiving the vehicle but were closer to naïve control levels in TBI mice receiving above doses of hMSC-EVs. Notably, improved BDNF concentration observed in TBI mice receiving hMSC-EVs in the acute phase was sustained in the chronic phase of TBI. Thus, a single IN dose of hMSC-EVs at 90 min post-TBI can ease TBI-induced declines in the BDNF-ERK-CREB signaling, hippocampal neurogenesis, and synapses.
RESUMO
Cancer antigen 125 (CA125), also referred to as mucin 16, is expressed under both normal and pathological conditions and the complexity of its structure indicates multifunctionality, i.e. both the protein and carbohydrate parts may be involved in diverse interactions at different levels of cell and tissue organization. Its biological role is not understood, but involvement in immune response modulation and influence on cell adhesion have been speculated. This study aimed at isolation and characterization of endogenous ligands for CA125 as an initial step in gaining insight into its activity. A CA125-reactive fraction was separated from human placental extract by affinity chromatography. The isolated preparation was characterized by SDS-PAGE, immunoblotting, peptide mass fingerprinting and binding assay. The CA125-reactive fraction from placental extract was identified as carbohydrate-binding IgG. The glycan composition of inhibitors of carbohydrate-binding pointed to sialic acid as one determinant for recognition but indicated that sialylation was not alone and that glycotopes containing galactose, N-acetylgalactosamine and N-acetylglucosamine were also important. CA125-reactive IgG could be selectively enriched using fetuin as the ligand and represents a distinct IgG subfraction differing from abundant natural carbohydrate-binding antibodies. Taking advantage of the particular properties of ligands for CA125 may have biomedical potential for use as biological modifiers or delivery agents and have an impact beyond pregnancy, since many immunoregulatory molecular pathways are common to embryonic development and malignant transformation.
Assuntos
Antígeno Ca-125/metabolismo , Imunoglobulina G/metabolismo , Placenta/imunologia , Placenta/metabolismo , Proteínas da Gravidez/imunologia , Proteínas da Gravidez/metabolismo , Animais , Metabolismo dos Carboidratos , Proteínas de Transporte/imunologia , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Cromatografia de Afinidade , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Imunoglobulina G/isolamento & purificação , Técnicas In Vitro , Ligantes , Camundongos , Microscopia de Fluorescência , Mucinas/metabolismo , Gravidez , Proteínas da Gravidez/isolamento & purificação , Ligação Proteica , Coelhos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Background: Extracellular vesicles (EVs), released from the plasma membrane or intracellular compartments, have a specific composition related to their parent cells, but they can, additionally, be modified by the extracellular environment. Although glycans are known to contribute to EV composition and may have biomedical importance as biomarkers and recognition signals, they have not been extensively investigated. In this study, seminal prostasomes, i.e. EVs from seminal plasma (SP) of normo- and oligozoospermic men, were analyzed in order to detect possible changes in their surface glycans under altered physiological conditions. Methods: Prostasomes were isolated from pooled SP by differential centrifugation and gel filtration, followed by glycobiochemical characterization using lectin/immune-transmission microscopy and ion-exchange chromatography. Results: Within the frame of overall similarity in protein composition, surface glycans specifically contributed to the differences between the examined groups of prostasomes in terms of presentation of sialylated and mannosylated moieties. These changes did not affect their anti-oxidative capacity, but implied a possible influence on the accessibility of galectin-3 to its ligands on the prostasomal surface. Conclusions: Subtle differences in the presentation of surface molecules may be helpful for differentiation among vesicles sharing the same physical properties. In addition, this may point to some unexpected regulatory mechanisms of interaction of distinct populations of vesicles with their binding partners.
Assuntos
Oligospermia/metabolismo , Oligospermia/fisiopatologia , Polissacarídeos/química , Próstata/metabolismo , Sêmen/metabolismo , Espermatozoides/metabolismo , Antioxidantes/química , Biomarcadores/metabolismo , Centrifugação , Cromatografia em Gel , Cromatografia por Troca Iônica , Galectina 3/química , Glicosilação , Humanos , Ligantes , Masculino , Microscopia Eletrônica de Transmissão , Ácido N-Acetilneuramínico/química , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismoRESUMO
A microscale procedure for the isolation of transferrin directly from human serum (hTf) is described in this study. The protocol is based on three precipitation steps without application of chromatography. It lasts 90min with the initial sample volume of 250µL. The yield of the isolated hTf is 58%, which is considerable in biochemical terms. The purity of the isolated hTf is 97%, as assessed by three methods: electrophoresis followed by protein staining, immunoblotting and HPLC. Immunoblotting with antibodies against other major serum proteins indicated that isolated hTf does not contain albumin, immunoglobulin G or alpha-2-macroglobulin. Lectin dot-blot demonstrated that isolated hTf preserved its glycan moieties. Fluorescent emission spectroscopy of the isolated hTf has shown no changes in tertiary structure. Isolated hTf was approximately 26% saturated with iron ion, which is comparable to physiological value (although a degree of saturation decreases to some extent during isolation procedure). Finally, co-immunoprecipitation experiment confirmed that isolated hTf retained its ligand characteristics crucial for the ligand-receptor type of interaction with the hTf receptor. To conclude, the procedure described in this work, is time and cost-effective, allows multiple sample handling and provides high-purity hTf isolate with preserved structural and functional properties.
Assuntos
Imunoprecipitação/métodos , Soro/química , Transferrina/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Glicosilação , Humanos , Receptores da Transferrina/metabolismo , Transferrina/análise , Transferrina/metabolismoRESUMO
Despite numerous studies, isolating pure preparations of extracellular vesicles (EVs) has proven challenging. Here, we compared ion-exchange chromatography (IEC) to the widely used sucrose density gradient (SDG) centrifugation method for the purification of EVs. EVs in bulk were isolated from pooled normal human amniotic fluid (AF) by differential centrifugation followed by IEC or sucrose density gradient separation. The purity of the isolated EVs was evaluated by electrophoresis and lectin blotting/immuno blotting to monitor the distribution of total proteins, different EVs markers, and selected N-glycans. Our data showed efficient separation of negatively charged EVs from other differently charged molecules, while comparative profiling of EVs using SDG centrifugation confirmed anion-exchange chromatography is advantageous for EV purification. Finally, although this IEC-based method was validated using AF, the approach should be readily applicable to isolation of EVs from other sources as well.
Assuntos
Líquido Amniótico/química , Cromatografia por Troca Iônica/métodos , Vesículas Extracelulares/química , Feminino , Humanos , Polissacarídeos/análise , Gravidez , Proteínas/análiseRESUMO
CA125, a coelomic epithelium-related antigen, is expressed in both normal and pathological conditions. In this study, we compared the glycosylation of CA125 antigen from amniotic fluid and the ovarian carcinoma cell line OVCAR-3, in order to detect possible differences as a specific marker of their origin. Antigens from both sources were radiolabelled and subsequently subjected to the affinity chromatography, using plant lectins differing in carbohydrate specificity as ligands. A common chromatographic scheme was applied to all columns, i.e. they were eluted with: a) washing buffer to wash out non-bound and low-affinity bound fractions, b) a solution of inhibitory sugar and c) a low pH buffer, to release the high affinity bound fractions. CA125 antigen from each source was found to be heterogeneous in respect to the existence of multiple glycoforms, with O-linked glycan chains predominating. However, the binding patterns of both N- and O-linked glycan-reactive lectins indicated distinct differences in carbohydrate composition between CA125 antigen isolated from amniotic fluid and OVCAR-3 cell line. The observed specificites of CA125-oligosaccharide chains might be of special importance from the biomedical aspect, in terms of their possible use for clinical evaluation of gynecological functions in health and disease.