Apolipoprotein E imbalance in the cerebrospinal fluid of Alzheimer's disease patients.

OBJECTIVE: The purpose of this study was to examine the levels of cerebrospinal fluid (CSF) apolipoprotein E (apoE) species in Alzheimer's disease (AD) patients. METHODS: We analyzed two CSF cohorts of AD and control individuals expressing different APOE genotypes. Moreover, CSF samples from the TgF344-AD rat model were included. Samples were run in native- and SDS-PAGE under reducing or non-reducing conditions (with or without ß-mercaptoethanol). Immunoprecipitation combined with mass spectrometry or western blotting analyses served to assess the identity of apoE complexes. RESULTS: In TgF344-AD rats expressing a unique apoE variant resembling human apoE4, a ~35-kDa apoE monomer was identified, increasing at 16.5 months compared with wild-types. In humans, apoE isoforms form disulfide-linked dimers in CSF, except apoE4, which lacks a cysteine residue. Thus, controls showed a decrease in the apoE dimer/monomer quotient in the APOE ε3/ε4 group compared with ε3/ε3 by native electrophoresis. A major contribution of dimers was found in APOE ε3/ε4 AD cases, and, unexpectedly, dimers were also found in ε4/ε4 AD cases. Under reducing conditions, two apoE monomeric glycoforms at 36 kDa and at 34 kDa were found in all human samples. In AD patients, the amount of the 34-kDa species increased, while the 36-kDa/34-kDa quotient was lower compared with controls. Interestingly, under reducing conditions, a ~100-kDa apoE complex, the identity of which was confirmed by mass spectrometry, also appeared in human AD individuals across all APOE genotypes, suggesting the occurrence of aberrantly resistant apoE aggregates. A second independent cohort of CSF samples validated these results. CONCLUSION: These results indicate that despite the increase in total apoE content the apoE protein is altered in AD CSF, suggesting that function may be compromised.

Dense core vesicle markers in CSF and cortical tissues of patients with Alzheimer's disease.

BACKGROUND: New fluid biomarkers for Alzheimer's disease (AD) that reveal synaptic and neural network dysfunctions are needed for clinical practice and therapeutic trial design. Dense core vesicle (DCV) cargos are promising cerebrospinal fluid (CSF) indicators of synaptic failure in AD patients. However, their value as biomarkers has not yet been determined. METHODS: Immunoassays were performed to analyze the secretory proteins prohormone convertases PC1/3 and PC2, carboxypeptidase E (CPE), secretogranins SgIII and SgII, and Cystatin C in the cerebral cortex (n = 45, provided by Bellvitge University Hospital) and CSF samples (n = 66, provided by The Sant Pau Initiative on Neurodegeneration cohort) from AD patients (n = 56) and age-matched controls (n = 55). RESULTS: In AD tissues, most DCV proteins were aberrantly accumulated in dystrophic neurites and activated astrocytes, whereas PC1/3, PC2 and CPE were also specifically accumulated in hippocampal granulovacuolar degeneration bodies. AD individuals displayed an overall decline of secretory proteins in the CSF. Interestingly, in AD patients, the CSF levels of prohormone convertases strongly correlated inversely with those of neurodegeneration markers and directly with cognitive impairment status. CONCLUSIONS: These results demonstrate marked alterations of neuronal-specific prohormone convertases in CSF and cortical tissues of AD patients. The neuronal DCV cargos are biomarker candidates for synaptic dysfunction and neurodegeneration in AD.

Dye-doped biodegradable nanoparticle SiO2 coating on zinc- and iron-oxide nanoparticles to improve biocompatibility and for in vivo imaging studies.

In vivo imaging and therapy represent one of the most promising areas in nanomedicine. Particularly, the identification and localization of nanomaterials within cells and tissues are key issues to understand their interaction with biological components, namely their cell internalization route, intracellular destination, therapeutic activity and possible cytotoxicity. Here, we show the development of multifunctional nanoparticles (NPs) by providing luminescent functionality to zinc and iron oxide NPs. We describe simple synthesis methods based on modified Stöber procedures to incorporate fluorescent molecules on the surface of oxide NPs. These procedures involve the successful coating of NPs with size-controlled amorphous silica (SiO2) shells incorporating standard chromophores like fluorescein, rhodamine B or rhodamine B isothiocyanate. Specifically, spherical Fe3O4 NPs with an average size of 10 nm and commercial ZnO NPs (ca. 130 nm), both coated with an amorphous SiO2 shell of ca. 15 and 24 nm thickness, respectively, are presented. The magnetic nanoparticles, with a major presence of magnetite, show negligible coercitivity. Hence, interactions (dipolar) are very weak and the cores are in the superparamagnetic regime. Spectroscopic measurements confirm the presence of fluorescent molecules within the SiO2 shell, making these hybrid NPs suitable for bioimaging. Thus, our coating procedures improve NP dispersibility in physiological media and allow the identification and localization of intracellular ZnO and Fe3O4 NPs using confocal microscopy imaging preserving the fluorescence of the NP. We demonstrate how both Fe3O4 and ZnO NPs coated with luminescent SiO2 are internalized and accumulated in the cell cytoplasm after 24 hours. Besides, the SiO2 shell provides a platform for further functionalization that enables the design of targeted therapeutic strategies. Finally, we studied the degradation of the shell in different physiological environments, pointing out that the SiO2 coating is stable enough to reach the target cells maintaining its original structure. Degradation took place only 24 hours after exposure to different media.

Exploring tumourigenic potential of the parasite Anisakis: a pilot study.

Anisakiasis is a global disease caused by consumption of raw or lightly cooked fish parasitised with Anisakis spp. third-stage larvae. Cases in the literature show colocalised anisakiasis and colorectal cancer, and the incidental finding of Anisakis larvae at the tumour site was reported. Data from our group suggested an epidemiological link between previous infection and gastrointestinal cancer. Furthermore, it has recently been reported that Anisakis products lead to inflammation and DNA damage. Based on these facts, the aim was to investigate whether Anisakis antigens are able to induce changes in the proliferation of epithelial cells in vitro or in the expression of serum microRNA (miRNA) in Sprague-Dawley rats. Anisakis complete extract (CE) induced increases in cell proliferation and decreases in apoptosis compared with nontreated cells, which resulted in a significant increase in the absolute number of viable cells at 48 h of exposure (P < .05). Furthermore, the miRNAs mmu-miR-1b-5p and mmu-miR-10b-5p (a cancer-related miRNA) were significantly decreased (P < .05) in sera from the rats inoculated with Anisakis CE, compared with control rats inoculated with saline. Additionally, based on their relative quantification values, four other cancer-related miRNAs were considered to be differently expressed, rno-miR-218a-5p and mmu-miR-224-5p (decreased) and rno-miR-125a-3p and rno-miR-200c-3p (increased). Anisakis CE was able to induce changes both in epithelial cells in vitro and in an animal model. The results obtained with Anisakis CE, in terms of increasing cell proliferation, decreasing apoptosis and inducing changes in the expression of serum cancer-related miRNAs in rats, suggest that Anisakis could have tumourigenic potential.

Cyclic AMP signaling restricts activation and promotes maturation and antioxidant defenses in astrocytes.

BACKGROUND: cAMP signaling produces dramatic changes in astrocyte morphology and physiology. However, its involvement in phenotype acquisition and the transcriptionally mediated mechanisms of action are largely unknown. RESULTS: Here we analyzed the global transcriptome of cultured astroglial cells incubated with activators of cAMP pathways. A bulk of astroglial transcripts, 6221 annotated genes, were differentially regulated by cAMP signaling. cAMP analogs strongly upregulated genes involved in typical functions of mature astrocytes, such as homeostatic control, metabolic and structural support to neurons, antioxidant defense and communication, whereas they downregulated a considerable number of proliferating and immaturity-related transcripts. Moreover, numerous genes typically activated in reactive cells, such as scar components and immunological mediators, were repressed by cAMP. GSEA analysis contrasting gene expression profiles with transcriptome signatures of acutely isolated astrocytes and in situ evaluation of protein levels in these cells showed that cAMP signaling conferred mature and in vivo-like transcriptional features to cultured astrocytes. CONCLUSIONS: These results indicate that cAMP signaling is a key pathway promoting astrocyte maturation and restricting their developmental and activation features. Therefore, a positive modulation of cAMP signaling may promote the normal state of differentiated astrocytes and favor the protection and function of neuronal networks.

A novel role for an RCAN3-derived peptide as a tumor suppressor in breast cancer.

The members of the human regulators of calcineurin (RCAN) protein family are endogenous regulators of the calcineurin (CN)-cytosolic nuclear factor of activated T-cells (NFATc) pathway activation. This function is explained by the presence of a highly conserved calcipressin inhibitor of calcineurin (CIC) motif in RCAN proteins, which has been shown to compete with NFATc for the binding to CN and therefore are able to inhibit NFATc dephosphorylation and activation by CN. Very recently, emerging roles for NFATc proteins in transformation, tumor angiogenesis and metastasis have been described in different cancer cell types. In this work, we report that the overexpression of RCAN3 dramatically inhibits tumor growth and tumor angiogenesis in an orthotopic human breast cancer model. We suggest that RCAN3 exerts these effects in a CN-dependent manner, as mutation of the CIC motif in RCAN3 abolishes the tumor suppressor effect. Moreover, the expression of the EGFP-R3(178-210) peptide, spanning the CIC motif of RCAN3, is able to reproduce all the antitumor effects of RCAN3 full-length protein. Finally, we show that RCAN3 and the EGFP-R3(178-210) peptide inhibit the CN-NFATc signaling pathway and the induction of the NFATc-dependent gene cyclooxygenase-2. Our work suggests that the EGFP-R3(178-210) peptide possess potent tumor suppressor properties and therefore constitutes a novel lead for the development of potent and specific antitumoral agents. Moreover, we propose the targeting of the CN-NFATc pathway in the tumor cells constitutes an effective way to hamper tumor progression by impairing the paracrine network among tumor, endothelial and polymorphonucleated cells.

Serotonin skews human macrophage polarization through HTR2B and HTR7.

Besides its role as a neurotransmitter, serotonin (5-hydroxytryptamine, 5HT) regulates inflammation and tissue repair via a set of receptors (5HT(1-7)) whose pattern of expression varies among cell lineages. Considering the importance of macrophage polarization plasticity for inflammatory responses and tissue repair, we evaluated whether 5HT modulates human macrophage polarization. 5HT inhibited the LPS-induced release of proinflammatory cytokines without affecting IL-10 production, upregulated the expression of M2 polarization-associated genes (SERPINB2, THBS1, STAB1, COL23A1), and reduced the expression of M1-associated genes (INHBA, CCR2, MMP12, SERPINE1, CD1B, ALDH1A2). Whereas only 5HT(7) mediated the inhibitory action of 5HT on the release of proinflammatory cytokines, both 5HT(2B) and 5HT(7) receptors mediated the pro-M2 skewing effect of 5HT. In fact, blockade of both receptors during in vitro monocyte-to-macrophage differentiation preferentially modulated the acquisition of M2 polarization markers. 5HT(2B) was found to be preferentially expressed by anti-inflammatory M2(M-CSF) macrophages and was detected in vivo in liver Kupffer cells and in tumor-associated macrophages. Therefore, 5HT modulates macrophage polarization and contributes to the maintenance of an anti-inflammatory state via 5HT(2B) and 5HT(7), whose identification as functionally relevant markers for anti-inflammatory/homeostatic human M2 macrophages suggests their potential therapeutic value in inflammatory pathologies.

Hypodermal decidualized endometrioma with aberrant cytokeratin expression. A lesion mimicking malignancy.

Decidualized endometrioma is a pseudoneoplastic lesion that may appear as a solitary nodule in the hypodermis, simulate a malignant epithelioid tumor, and can represent a diagnostic challenge. A 36-year-old woman delivered a full-term baby by cesarean. At the immediate puerperium, she complained of a subcutaneous nodule measuring 2.5 cm, underneath a previous caesarean scar from the former full-term delivery 3 years earlier. Histologic features included a nodular growth pattern of large monomorphic epithelioid cells showing diffuse positivity for cytokeratin (AE1/AE3, 18), human placental lactogen, and CD10 and focal positivity for inhibin alpha. The main differential diagnoses include trophoblastic neoplasia and deciduoid mesothelioma. Good clinicopathological correlation is essential for the correct diagnosis. Immunohistochemical stains can be misleading. An important clue is the combination of large decidualized cells and lumens lined by flat or low cuboidal cells that are atrophic endometrial glands. This lesion has a benign behavior.

Dendritic cell-specific ICAM-3-grabbing nonintegrin expression on M2-polarized and tumor-associated macrophages is macrophage-CSF dependent and enhanced by tumor-derived IL-6 and IL-10.

Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN; CD209) is a human pathogen-attachment C-type lectin with no obvious murine ortholog and for which ligation leads to enhanced anti-inflammatory cytokine release and altered proinflammatory cytokine production. Although induced by IL-4 in monocytes and considered as a DC marker, DC-SIGN expression on human APCs under homeostatic conditions is so far unexplained. We report in this study that M-CSF enhances DC-SIGN expression on in vitro derived anti-inflammatory macrophages and that M-CSF mediates the induction of DC-SIGN by fibroblast- and tumor cell-conditioned media. The M-CSF-inducible DC-SIGN expression along monocyte-to-macrophage differentiation is dependent on JNK and STAT3 activation, potentiated by STAT3-activating cytokines (IL-6, IL-10), and abrogated by the M1-polarizing cytokine GM-CSF. In pathological settings, DC-SIGN expression is detected in tumor tissues and on ex vivo-isolated CD14(+) CD163(+) IL-10-producing tumor-associated macrophages. Importantly, DC-SIGN Abs reduced the release of IL-10 from macrophages exposed to Lewis(x)-expressing SKBR3 tumor cells. These results indicate that DC-SIGN is expressed on both wound-healing (IL-4-dependent) and regulatory (M-CSF-dependent) alternative (M2) macrophages and that DC-SIGN expression on tumor-associated macrophages might help tumor progression by contributing to the maintenance of an immunosuppressive environment.

Content and traffic of taurine in hippocampal reactive astrocytes.

Taurine is one of the most abundant free amino acids in the mammalian central nervous system, where it is crucial to proper development. Moreover, taurine acts as a neuroprotectant in various diseases; in epilepsy, for example, it has the capacity to reduce or abolish seizures. In the present study, taurine levels has been determine in mice treated with Kainic Acid (KA) and results showed an increase of this amino acid in hippocampus but not in whole brain after 3 and 7 days of KA treatment. This increase occurs when gliosis was observed. Moreover, taurine transporter (TAUT) was found in astrocytes 3 and 7 days after KA treatment, together with an increase in cysteine sulfinic acid decarboxylase (csd) mRNA, that codifies for the rate-limiting enzyme of taurine synthesis, in the hippocampus at the same times after KA treatment. Glial cultures enriched in astrocytes were developed to demonstrate that these cells are responsible for changes in taurine levels after an injury to the brain. The cultures were treated with proinflammatory cytokines to reproduce gliosis. In this experimental model, an increase in the immunoreactivity of GFAP was observed, together with an increase in CSD and taurine levels. Moreover, an alteration in the taurine uptake-release kinetics was detected in glial cells treated with cytokine. All data obtained indicate that astrocytes could play a key role in taurine level changes induced by neuronal damage. More studies are, therefore, needed to clarify the role taurine has in relation to neuronal death and repair.

Splanchnic Th(2) and Th(1) cytokine redistribution in microsurgical cholestatic rats.

BACKGROUND: Long-term extrahepatic cholestasis in the rat induces ductular proliferation and fibrosis in the liver, portal hypertension, splenomegaly, portosystemic collateral circulation, and ascites. These splanchnic alterations could have an inflammatory pathophysiology. MATERIAL AND METHODS: We measured serum levels of hepatobiliary injury markers and the acute phase proteins, alpha-1-major acid protein (alpha(1)-MAP) and alpha-1-acid glycoprotein (alpha(1)-GPA) in rats 6 wk after microsurgical extrahepatic cholestasis. We also assayed Th(1) (TNF-alpha and IL-1beta) and Th(2) (IL-4 and IL-10) cytokine levels in the liver, ileum, spleen, and mesenteric lymph complex by enzyme-linked immunosorbent assay (ELISA) techniques. Liver fibrosis was measured by Sirius red stain and by using an image system computer-assisted method and mast cell liver infiltration by Giemsa stain. RESULTS: The cholestatic rats showed an increase (P<0.001) in serum levels of bile acids, total and direct bilirubin, AST, ALT, AST/ALT index, gamma-GT, alkaline phosphatase, alpha(1)- MAP, alpha(1)-GPA, and LDH (P<0.05) in relation to sham-operated rats. TNF-alpha, IL-1beta, IL-4, and IL-10 increased in the ileum (P<0.01) and mesenteric lymph complex (P<0.001), and decreased in the liver (P<0.001). A marked bile proliferation associated with fibrosis (P<0.001) and mast cell infiltration was also shown in the liver of cholestatic rats. CONCLUSION: The splanchnic redistribution of cytokines, with an increase of Th(1) and Th(2) production in the small bowel and in the mesenteric lymph complex, supports the key role of inflammatory mechanisms in rats with secondary biliary fibrosis.

Folate receptor beta is expressed by tumor-associated macrophages and constitutes a marker for M2 anti-inflammatory/regulatory macrophages.

Macrophage activation comprises a continuum of functional states critically determined by cytokine microenvironment. Activated macrophages have been functionally grouped according to their response to pro-Th1/proinflammatory stimuli [lipopolysaccharide, IFNgamma, granulocyte macrophage colony-stimulating factor (GM-CSF); M1] or pro-Th2/anti-inflammatory stimuli [interleukin (IL)-4, IL-10, M-CSF; M2]. We report that folate receptor beta (FRbeta), encoded by the FOLR2 gene, is a marker for macrophages generated in the presence of M-CSF (M2), but not GM-CSF (M1), and whose expression correlates with increased folate uptake ability. The acquisition of folate uptake ability by macrophages is promoted by M-CSF, maintained by IL-4, prevented by GM-CSF, and reduced by IFNgamma, indicating a link between FRbeta expression and M2 polarization. In agreement with in vitro data, FRbeta expression is detected in tumor-associated macrophages (TAM), which exhibit an M2-like functional profile and exert potent immunosuppressive functions within the tumor environment. FRbeta is expressed, and mediates folate uptake, by CD163(+) CD68(+) CD14(+) IL-10-producing TAM, and its expression is induced by tumor-derived ascitic fluid and conditioned medium from fibroblasts and tumor cell lines in an M-CSF-dependent manner. These results establish FRbeta as a marker for M2 regulatory macrophage polarization and indicate that folate conjugates of therapeutic drugs are a potential immunotherapy tool to target TAM.

Regulation of exocytotic protein expression and Ca2+-dependent peptide secretion in astrocytes.

Vesicular transmitter release from astrocytes influences neuronal development, function and plasticity. However, secretory pathways and the involved molecular mechanisms in astroglial cells are poorly known. In this study, we show that a variety of SNARE and Munc18 isoforms are expressed by cultured astrocytes, with syntaxin-4, Munc18c, SNAP-23 and VAMP-3 being the most abundant variants. Exocytotic protein expression was differentially regulated by activating and differentiating agents. Specifically, proteins controlling Ca(2+)-dependent secretion in neuroendocrine cells were up-regulated after long-term 8Br-cAMP administration in astrocytes, but not by proinflammatory cytokines. Moreover, 8Br-cAMP treatment greatly increased the cellular content of the peptidic vesicle marker secretogranin-2. Release assays performed on cAMP-treated astrocytes showed that basal and stimulated secretogranin-2 secretion are dependent on [Ca(2+)](i). As shown release of the chimeric hormone ANP.emd from transfected cells, cAMP-induced differentiation in astrocytes enhances Ca(2+)-regulated peptide secretion. We conclude that astroglial cells display distinctive molecular components for exocytosis. Moreover, the regulation of both exocytotic protein expression and Ca(2+)-dependent peptide secretion in astrocytes by differentiating and activating agents suggest that glial secretory pathways are adjusted in different physiological states.

The pathogen receptor liver and lymph node sinusoidal endotelial cell C-type lectin is expressed in human Kupffer cells and regulated by PU.1.

UNLABELLED: Human LSECtin (liver and lymph node sinusoidal endothelial cell C-type lectin, CLEC4G) is a C-type lectin encoded within the L-SIGN/DC-SIGN/CD23 gene cluster. LSECtin acts as a pathogen attachment factor for Ebolavirus and the SARS coronavirus, and its expression can be induced by interleukin-4 on monocytes and macrophages. Although reported as a liver and lymph node sinusoidal endothelial cell-specific molecule, LSECtin could be detected in the MUTZ-3 dendritic-like cell line at the messenger RNA (mRNA) and protein level, and immunohistochemistry analysis on human liver revealed its presence in Kupffer cells coexpressing the myeloid marker CD68. The expression of LSECtin in myeloid cells was further corroborated through the analysis of the proximal regulatory region of the human LSECtin gene, whose activity was maximal in LSECtin+ myeloid cells, and which contains a highly conserved PU.1-binding site. PU.1 transactivated the LSECtin regulatory region in collaboration with hematopoietic-restricted transcription factors (Myb, RUNX3), and was found to bind constitutively to the LSECtin proximal promoter. Moreover, knockdown of PU.1 through the use of small interfering RNA led to a decrease in LSECtin mRNA levels in THP-1 and monocyte-derived dendritic cells, thus confirming the involvement of PU.1 in the myeloid expression of the lectin. CONCLUSION: LSECtin is expressed by liver myeloid cells, and its expression is dependent on the PU.1 transcription factor.

Molecular pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts: mutations in MLC1 cause folding defects.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy, most often caused by mutations in the MLC1 gene. MLC1 is an oligomeric plasma membrane (PM) protein of unknown function expressed mainly in glial cells and neurons. Most disease-causing missense mutations dramatically reduced the total and PM MLC1 expression levels in Xenopus oocytes and mammalian cells. The impaired expression of the mutants was verified in primary cultures of rat astrocytes, as well as human monocytes, cell types that endogenously express MLC1, demonstrating the relevance of the tissue culture models. Using a combination of biochemical, pharmacological and imaging methods, we also demonstrated that increased endoplasmatic reticulum-associated degradation and endo-lysosomal-associated degradation can contribute to the cell surface expression defect of the mutants. Based on these results, we suggest that MLC1 mutations reduce protein levels in vivo. Since the expression defect of the mutants could be rescued by exposing the mutant-protein expressing cells to low temperature and glycerol, a chemical chaperone, we propose that MLC belongs to the class of conformational diseases. Therefore, we suggest the use of pharmacological strategies that improve MLC1 expression to treat MLC patients.

Expression patterns of MLC1 protein in the central and peripheral nervous systems.

Mutations in MLC1 cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), a disorder characterized clinically by macrocephaly, deterioration of motor functions, epilepsy and mental decline. Recent studies have detected MLC1 mRNA and protein in astroglial processes. In addition, our group previously reported MLC1 expression in some neurons in the adult mouse brain. Here we performed an exhaustive study of the expression pattern of MLC1 in the developing mouse brain by means of optic and electron microscopy. In the central nervous system, MLC1 was detected mainly in axonal tracts early in development. In addition, MLC1 was also observed in the peripheral nervous system and in several sensory epithelia, as retina or saccula maculae. Post-embedding immunogold experiments indicated that MLC1 is localized in astrocyte-astrocyte junctions, but not in the perivascular membrane, indicating that MLC1 is not a component of the dystrophin-glycoprotein complex. In neurons, MLC1 is located at the plasma membrane and vesicular structures. Our data provide a mouse MLC1 expression map that could be useful to understand the phenotype of MLC patients, and suggested that MLC disease is caused by an astrocytic and a neuronal dysfunction.

Cloning and characterisation of the Anisakis simplex allergen Ani s 4 as a cysteine-protease inhibitor.

Anisakis simplex is a nematode that can parasitise humans who eat raw or undercooked fish containing live L3s. Larvae invading the gastrointestinal mucosa excrete/secrete proteins implicated in the pathogenesis of anisakiasis that can induce IgE mediated symptoms. Misdiagnosis of anisakiasis, due to cross-reactivity, makes it necessary to develop new diagnostic tools. Recombinant allergens have proved to be useful for diagnosis of other parasitoses. Among the Anisakis allergens, Ani s 4 was considered to be a good potential diagnostic protein because of its heat resistance and its importance in the clinical history of sensitised patients. Therefore, the objective of this study was to clone and characterise the cDNA encoding this allergen. The Ani s 4 mRNA sequence was obtained using a PCR-based strategy. The Ani s 4 amino acid sequence contained the characteristic domains of cystatins. Mature recombinant Ani s 4 was expressed in a bacterial system as a His-tagged soluble protein. The recombinant Ani s 4 inhibited the cleavage of a peptide substrate by papain with a Ki value of 20.6 nM. Immunobloting, ELISA, a commercial fluorescence-enzyme-immunoassay and a basophil activation test were used to study the allergenic properties of rAni s 4, demonstrating that the recombinant allergen contained the same IgE epitopes as the native Ani s 4, and that it was a biologically active allergen since it activated basophils from patients with allergy to A. simplex in a specific concentration-dependent manner. Ani s 4 was localised by immunohistochemical methods, using a polyclonal anti-Ani s 4 anti-serum, in both the secretory gland and the basal layer of the cuticle of A. simplex L3. In conclusion, we believe that Ani s 4 is the first nematode cystatin that is a human allergen. The resulting rAni s 4 retains all allergenic properties of the natural allergen, and can therefore be used in immunodiagnosis of human anisakiasis.