Weighted gene co-expression network analysis reveals immune evasion related genes in Echinococcus granulosus sensu stricto.

Cystic echinococcosis (CE) is a zoonotic disease caused by the tapeworm Echinococcus granulosus sensu lato (s.l). In the intermediate host, this disease is characterized by the growth of cysts in viscera such as liver and lungs, inside of which the parasite develops to the next infective stage known as protoscoleces. There are records that the infected viscera affect the development and morphology of E. granulosus s.l. protoscolex in hosts such as buffalo or humans. However, the molecular mechanisms that drive these differences remains unknown. Weighted gene co-expression network analysis (WGCNA) using a set of RNAseq data obtained from E. granulosus sensu stricto (s.s.) protoscoleces found in liver and lung cysts reveals 34 modules in protoscoleces of liver origin, of which 12 have differential co-expression from protoscoleces of lung origin. Three of these twelve modules contain hub genes related to immune evasion: tegument antigen, tegumental protein, ubiquitin hydrolase isozyme L3, COP9 signalosome complex subunit 3, tetraspanin CD9 antigen, and the methyl-CpG-binding protein Mbd2. Also, two of the twelve modules contain only hypothetical proteins with unknown orthology, which means that there are a group of unknown function proteins co-expressed inside the protoscolex of liver CE cyst origin. This is the first evidence of gene expression differences in protoscoleces from CE cysts found in different viscera, with co-expression networks that are exclusive to protoscoleces from liver CE cyst samples. This should be considered in the control strategies of CE, as intermediate hosts can harbor CE cysts in liver, lungs, or both organs simultaneously.

Transcriptome analysis of Echinococcus granulosus sensu stricto protoscoleces reveals differences in immune modulation gene expression between cysts found in cattle and sheep.

Cystic Echinococcosis (CE), a zoonotic parasitic disease, is caused by the cestode Echinococcus granulosus sensu lato. CE inflicts severe damage in cattle, sheep, and human hosts worldwide. Fertile CE cysts are characterized by the presence of viable protoscoleces. These parasite forms are studied with minimal contamination with host molecules. Hosts, cattle and sheep, show differences in their CE cyst fertility. The effect of the host in protoscolex transcriptome is not known. We genotyped and performed transcriptomic analysis on sheep protoscoleces obtained from liver and lung CE cysts. The transcriptomic data of Echinococcus granulosus sensu stricto protoscoleces from 6 lung CE cysts and 6 liver CE cysts were Collected. For host comparison analysis, 4 raw data files belonging to Echinococcus granulosus sensu stricto protoscoleces from cattle liver CE cysts were obtained from the NCBI SRA database. Principal component and differential expression analysis did not reveal any statistical differences between protoscoleces obtained from liver or lung cysts, either within the same sheep or different sheep hosts. Conversely, there are significant differences between cattle and sheep protoscolex samples. We found differential expression of immune-related genes. In cattle, 7 genes were upregulated in protoscoleces from liver cysts. In sheep, 3 genes were upregulated in protoscoleces from liver and lung CE cysts. Noteworthy, are the differential expression of antigen B, tegument antigen, and arginase-2 in samples obtained from sheep CE cysts, and basigin in samples from cattle CE cysts. These findings suggest that the host species is an important factor involved in the differential expression of immune related genes, which in turn is possibly related to the fertility of Echinococcus granulosus sensu stricto cysts.

Effectiveness of a New Recombinant antiGnRH Vaccine for Immunocastration in Bulls.

Castration by surgical techniques is common in livestock; however, post-surgery complications and concerns for animal wellbeing have created a need for new non-invasive alternatives. The objective of this study was to evaluate immunocastration in bulls using antigen GnRX G/Q; a recombinant peptide proved to be effective in laboratory and companion animals. A nine-month trial with 80 9-month-old Normand x Hereford bulls, kept in a pastured system, was conducted. The herd was divided in half with 40 bulls surgically castrated (SC) and 40 castrated by immunization against GnRH (IC). The antigen was injected on days 0 and 40 of the experiment. After the second dose, the IC group had elevated GnRH antibodies and decreased testosterone levels (below 5 ng/mL) that were maintained for 23 weeks. At slaughter on day 190, the immunocastrated group obtained a higher weight, hot carcass, and dressing percentage than the SC group. There was no difference in pH, color of meat, fat coverage, cooking loss, or tenderness between groups. The bulls showed no inflammatory reaction at the injection site or adverse side effects from the vaccine. Our results demonstrate that immunocastration with GnRX G/Q is an efficient and safe alternative to surgical castration in livestock. Additional work evaluating antigen effects over a longer period is needed to validate commercial viability.

Fasciola hepatica co-infection enhances Th1 immune response in the adventitial layer of non-fertile Echinococcus granulosus cysts.

Cystic echinococcosis is a widespread zoonosis caused by the larval stage of the tapeworm Echinococcus granulosus. In intermediary hosts, two types of echinococcal cysts can be found: fertile, which produce protoscoleces, the infective form of the parasite to dogs; and infertile, that do not present protoscoleces and therefore are not able to continue with the parasite life cycle. The adventitial layer, the local immune response against the cyst, plays an important role in cyst fertility. Grazing cattle can often feature Fasciola hepatica co-infection, a parasite known to modulate the host systemic immune response. In this work the cellular Th1/Th2 immune profiles were evaluated in the adventitial layer of fertile and non-fertile cysts with and without co-infection with Fasciola hepatica. Measuring with immunohistochemistry and qPCR in adventitial layer, we report that non-fertile cysts present higher levels of Th1 cytokines (IFN-γ (P < 0.0001) and TNF-α (P < 0.05)), and fertile cysts have higher levels of Th2 cytokines (IL-4 (P < 0.001)). Co-infection with Fasciola hepatica is associated with a decrease in the expression of IL-4 (P < 0.05) and an increase in the expression of IFN-γ (P < 0.0001) in the adventitial layer of fertile cysts. Non-fertile cysts were associated with higher levels of Th1 cytokines in the adventitial layer, with IFN-γ expression enhanced by F. hepatica co-infection (P < 0.0001), confirming that polyparasitism should be considered in the treatment and control of naturally infected cattle.

Lymphocyte Populations in the Adventitial Layer of Hydatid Cysts in Cattle: Relationship With Cyst Fertility Status and Fasciola Hepatica Co-Infection.

Cystic echinococcosis is a worldwide zoonosis caused by the cestode Echinococcus granulosus. Two types of hydatid cysts occur in intermediate hosts: fertile cysts that generate protoscoleces from the germinal layer of the cyst, and infertile cysts that do not produce protoscoleces and are unable to continue the life cycle of the parasite. The adventitial layer, a host-derived fibrous capsule surrounding the hydatid cyst, is suggested to play an important role in local immune regulation during infection and in fertility of the cysts. Fasciola hepatica, another important parasite of cattle, induces a characteristic Th2-like immune response that could modulate the immune response against E. granulosus. Natural co-infection of both parasites is common in cattle, but no reports describe the local immune response against E. granulosus with F. hepatica infection in the same host. This study analyzed the number and distribution of T and B cells in the adventitial layer of liver and lung cysts and the relationship with cyst fertility and F. hepatica co-infection. T lymphocytes were the predominant cell type in the adventitial layer of infertile hydatid cysts and were more numerous in infertile hydatid cysts. B lymphocyte numbers were not associated with hydatid cyst fertility. Mast cells were infrequent in the adventitial layer. The number of T and B cells was not associated with F. hepatica co-infection. The present study contributes to the understanding of local immune responses in bovine cystic echinococcosis.

Capacity to adhere to and invade human epithelial cells, as related to the presence of virulence genes in, motility of, and biofilm formation of Campylobacter jejuni strains isolated from chicken and cattle.

Campylobacter jejuni is a zoonotic pathogen transmitted through the "farm to fork" route. Outbreaks are generally associated with the consumption of chicken meat; however, dairy cows, birds, wild and domestic food animals, and pets are other important sources. Currently, there are not enough data comparing the virulence of strains isolated from these reservoirs. In this study, we compared C. jejuni strains isolated from broiler chickens and dairy cattle by determining their ability to adhere to and invade in vitro human colonic epithelial cells in the T84 cell line with their motility, formation of biofilms, and presence of eight virulence genes. A Wilcoxon Rank Sum test was performed to establish the relationship between presence of the studied genes and cellular invasion and adhesion, as well as differences between the animal species of origin of the isolate. A Spearman correlation was performed to assess the relationship between invasion and motility, along with invasion and biofilm generation. The virB11 gene was positively associated with the adherence capacity of the strains (mean difference = 0.21, p = 0.006), and strains isolated from chickens showed a significant difference for adherence compared with strains isolated from cattle (p = 0.0001). Our results indicate that strains of C. jejuni have a difference in their adherence capacity depending on the animal reservoir from which they came, with chicken isolates displaying higher virulence than dairy cattle isolates.

The immune profile induced is crucial to determine the effects of immunocastration over gonadal function, fertility, and GnRH-I expression.

PROBLEM: Immunocastration or vaccination against the GnRH-I hormone is a promising alternative to reproductive control in different animal species. Given the low immunogenicity of this hormone, the use of adjuvants becomes necessary. METHOD OF STUDY: This study evaluated the effects of three adjuvants that induce different immune response profiles over gonadal function, fertility, and expression of GnRH-I. Female mice (n = 6) were vaccinated at days 1 and 30 with a recombinant antigen for immunocastration and different adjuvants that induced preferentially Th1/Th2, Th2, and Th1 immune profiles. RESULTS: Th1/Th2 response is the most efficient to block reproductive activity in vaccinated animals, reducing the number of luteal bodies and pre-ovulatory follicles. Th2 and Th1/Th2 responses induced an increase in GnRH-I at the hypothalamus. CONCLUSION: The immune profile induced by different adjuvants is essential on the effects over fertility, gonadal function, and hypothalamic GnRH-I expression in immunocastrated animals.

Effectiveness of an immunocastration vaccine formulation to reduce the gonadal function in female and male mice by Th1/Th2 immune response.

Immunocastration has emerged as an alternative to surgical castration in different animal species. This study examined the effectiveness of a new vaccine formulation for immunocastration using the biopolymer chitosan as adjuvant. First, female and male mice (n = 4), in three subsequent experiments were vaccinated at Days 1 and 30 of the study, to determine the immune response profile and gonadal alterations due to immunization. The results demonstrated that the vaccine was able to elicit strong antibody responses against native GnRH hormone (P < 0.01), with a T helper (Th) 1/Th2 immune response profile. Along with this, a suppression of gonadal activity with a decrease of luteal bodies (1.08 ± 0.22 and 4.08 ± 0.39) and antral follicles (1.17 ± 0.32 and 4.5 ± 0.38) in the ovaries of immunized females and control, respectively, and a reduction of seminiferous tubules size (142.3 ± 5.58 mm and 198.0 ± 6.11 mm) and germinal cellular layers (3.58 ± 0.26 and 5.08 ± 0.29) of immunized males and control animals, respectively, were observed (P < 0.01). Then, in a study of long-term immune response due to vaccination in female and male mice (n = 4) from two subsequent experiments, a suppression of gonadal function and an induction of a Th1/Th2 immune response was also observed, determined by both, immunoglobulin and cytokine profiles, which lasted until the end of the study (7 months; P < 0.01). The findings of this study have demonstrated that vaccination with a new immunocastration vaccine inducing a Th1/Th2 immune response against GnRH (P < 0.01) elicit a decrease of gonadal function in male and female mice (P < 0.01). Owing to long-term duration of the antibody levels generated, this vaccine formulation appears as a promising alternative for immunocastration of several animal species where long-lasting reproductive block is needed.

Hepatogenic and neurogenic differentiation of bone marrow mesenchymal stem cells from abattoir-derived bovine fetuses.

BACKGROUND: Mesenchymal stem cells (MSC) are multipotent progenitor cells characterized by their ability to both self-renew and differentiate into tissues of mesodermal origin. The plasticity or transdifferentiation potential of MSC is not limited to mesodermal derivatives, since under appropriate cell culture conditions and stimulation by bioactive factors, MSC have also been differentiated into endodermal (hepatocytes) and neuroectodermal (neurons) cells. The potential of MSC for hepatogenic and neurogenic differentiation has been well documented in different animal models; however, few reports are currently available on large animal models. In the present study we sought to characterize the hepatogenic and neurogenic differentiation and multipotent potential of bovine MSC (bMSC) isolated from bone marrow (BM) of abattoir-derived fetuses. RESULTS: Plastic-adherent bMSC isolated from fetal BM maintained a fibroblast-like morphology under monolayer culture conditions. Flow cytometric analysis demonstrated that bMSC populations were positive for MSC markers CD29 and CD73 and pluripotency markers OCT4 and NANOG; whereas, were negative for hematopoietic markers CD34 and CD45. Levels of mRNA of hepatic genes α-fetoprotein (AFP), albumin (ALB), alpha1 antitrypsin (α1AT), connexin 32 (CNX32), tyrosine aminotransferase (TAT) and cytochrome P450 (CYP3A4) were up-regulated in bMSC during a 28-Day period of hepatogenic differentiation. Functional analyses in differentiated bMSC cultures evidenced an increase (P < 0.05) in albumin and urea production and glycogen storage. bMSC cultured under neurogenic conditions expressed NESTIN and MAP2 proteins at 24 h of culture; whereas, at 144 h also expressed TRKA and PrPC. Levels of MAP2 and TRKA mRNA were up-regulated at the end of the differentiation period. Conversely, bMSC expressed lower levels of NANOG mRNA during both hepatogenic and neurogenic differentiation processes. CONCLUSION: The expression patterns of linage-specific markers and the production of functional metabolites support the potential for hepatogenic and neurogenic differentiation of bMSC isolated from BM of abattoir-derived fetuses. The simplicity of isolation and the potential to differentiate into a wide variety of cell lineages lays the foundation for bMSC as an interesting alternative for investigation in MSC biology and eventual applications for regenerative therapy in veterinary medicine.

Bovine IgG subclasses and fertility of Echinococcus granulosus hydatid cysts.

Hydatidosis is an important zoonotic disease of worldwide distribution, causing important health problems to humans and major economical losses in infected livestock. Echinococcus granulosus, the etiological agent of hydatid disease, induces a humoral immune response in the intermediate host (human and herbivorous) against hydatid cyst antigens. Specifically, IgGs are found in the laminar and germinal layers and inside the lumen of fertile and infertile hydatid cysts. In the germinal layer of infertile cysts IgGs are found in an order of magnitude greater than in the germinal layer of fertile cysts; a fraction of those IgGs are associated with high affinity to germinal layer proteins, suggesting their binding to specific parasite antigens. We have previously shown that those immunoglobulins, bound with high affinity to the germinal layer of hydatid cysts, induce apoptosis leading to cyst infertility. In the present work the presence of IgG1 and IgG2 subclasses in the germinal layer of both fertile and infertile hydatid cysts is reported. IgG1 is the most relevant immunoglobulin subclass present in the germinal layer of infertile cysts and bound with high affinity to that parasite structure. Contrarily, though the IgG2 subclass was also found in the germinal and adventitial layers, those immunoglobulins show low affinity to parasite antigens. We propose that the binding of an IgG1 subclass to parasite antigens present in the germinal layer is involved in the mechanism of cyst infertility.

Inhibition of Egr-1 by siRNA in prostate carcinoma cell lines is associated with decreased expression of AP-1 and NF-κB.

Previous studies suggest that the effects of Egr-1 on tumorigenesis are critically dependent on the levels of Egr-1 and the cellular context. For this reason, we examined the effects of blocking the Egr-1 activity by a short interfering RNA (siRNA) against Egr-1 on the expression of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) signaling in the PC-3 and LNCaP prostate carcinoma cell lines. We observed that the reduction in expression of Egr-1 in PC-3 and LNCaP cells by effects of the siRNA vector resulted in lower cell viability and increased apoptosis at 24 and 120 h after transfection. This reduced cell viability correlated well with a reduced activity of the NF-κB and AP-1 factors. We analyzed the expression and activity of these factors and found that p65 and IκB but not p50 were reduced in the siRNA-treated cells. Similarly, we found that c-Fos but not c-Jun was reduced in the siRNA treated cells. These effects were translated to reduced transcriptional activity of NF-κB over cellular inhibitor of apoptosis (cIAP) and p21 genes, as assayed by RT-PCR and of AP-1 over a luciferase reporter activated by AP-1 response elements. Egr-1 was also found to interact directly with p65 and IκB members of the NF-κB family, thereby was able to regulate directly their activity by post-transcriptional effects. These results suggest that Egr-1 may promote prostate cancer development by modulating the activity of factors NF-κB and AP-1, which are involved in cell proliferation and apoptosis.

Bovine (Bos taurus) humoral immune response against Echinococcus granulosus and hydatid cyst infertility.

Echinococcus granulosus, the agent of hydatid disease, presents an indirect life cycle, with canines (mainly dogs) as definitive hosts, and herbivores and human as intermediary ones. In intermediary hosts fertile and infertile cysts develop, but only the first ones develop protoscoleces, the parasite form infective to definitive hosts. We report the presence of bovine IgGs in the germinal layer from infertile cysts (GLIC), in an order of magnitude greater than in the germinal layer from fertile cysts (GLFC). When extracted with salt solutions, bovine IgGs from GLIC are associated with low or with high affinity (most likely corresponding to non specific and antigen specific antibodies, respectively). Specific IgGs penetrate both the cells of the germinal layer and HeLa cultured cells and recognize parasitic proteins. These results, taken together with previous ones from our laboratory, showing induction of apoptosis in the germinal layer of infertile hydatid cysts, provide the first coherent explanation of the infertility process. They also offer the possibility of identifying the parasite antigens recognized, as possible targets for immune modulation.

Down-regulation of Egr-1 by siRNA inhibits growth of human prostate carcinoma cell line PC-3.

The inhibitory effect of a specific small EGR-1 interfering RNA (siRNA) on cell proliferation and the expression of EGR-1 in human prostate carcinoma cell lines PC-3 and LNCaP was investigated. To knockdown Egr-1 expression, a siRNA targeting against Egr-1 was synthesized and transfected into PC-3 and LNCaP cells. The down-regulation of Egr-1 expression at both mRNA and protein levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. The transcription activity was determined by luciferase expression. Cell proliferation inhibition rates were determined by soft agar and methyl thiazolyl tetrazolium (MTT) assay. The effect of Egr-1 siRNA on cell cycle distribution and cell apoptosis was determined by flow cytometry (FCM). RNA interference efficiently suppressed the Egr-1 expression in PC-3 and LNCaP cells. At 96 h after transfection, the expression inhibition rate was 44.52% at mRNA level detected by RT-PCR and 40.17% at protein level by Western blot analysis. The cell proliferation inhibition rates at 24, 48, 96 and 120 h after Egr-1 siRNA and non-silencing siRNA transfection, were 5, 25.06, 65.61 and 78.36%, respectively for PC-3 cells and 23, 40.3, 75.9 and 67.4%, respectively for LNCaP cells. The apoptosis rate was similar for both PC-3 and LNCaP and the number of cells was increased in G0/G1 phase from 38.2 to 88.6%, and decreased in S and G2/M phase at 96 h after transfection. Down-regulation of Egr-1 results in significant inhibition of tumor growth in vitro. The inhibition of Egr-1 expression can induce apoptosis of PC-3 cells. The use of Egr-1 siRNA deserves further investigation as a novel approach to cancer therapy.

Chitosan formulations improve the immunogenicity of a GnRH-I peptide-based vaccine.

Peptide vaccines using specific antigens with poor immunogenicity like GnRH-I are unable to develop an effective adaptive immune response and require the presence of adjuvants, essential to lymphocytic activation. Three chitosan formulations were evaluated for their ability as adjuvant of a poor immunogenic peptide vaccine against GnRH-I. Male Sprague-Dawley rats were immunized subcutaneously with recombinant His-GnRH-tandem-repeat peptide in high, low and phosphorylated high molecular weight chitosan solution at 0.5% (w/v). Freund's complete adjuvant was used as a positive control of immune response. Our results suggest that different chitosan formulations as adjuvant, with high or low viscosity degree allow inducing a high and persistent immune response against a poor immunogenic recombinant peptide. We found that the immune response was mediated by a increasing of IgG isotype 1, which were significantly greater than levels presented by the animals immunized with Freund's complete adjuvant. Nevertheless, chitosan with low molecular weight and highest acetylation degree was able to induce an immune response mediated by IgG isotype 2a. Additionally, high molecular weight phosphorylated chitosan, in which the phosphate groups were linked to N-acetyl-d-glucosamine unit, the immune response was reduced. All the immune responses obtained with chitosan as adjuvant were able to neutralize effectively the GnRH hormone proves by reducing of animal steroidogenesis and spermatogenesis demonstrating its capacity to improve immunogenicity in peptide vaccine.

Effect of leuprolide and cetrorelix on cell growth, apoptosis, and GnRH receptor expression in primary cell cultures from human prostate carcinoma.

Contradictory data have been reported regarding the effect of GnRH agonists and antagonists on cell growth and survival, using prostate cancer-derived cell lines expressing either endogenous or exogenous GnRH receptors. We addressed the issue studying the effect of leuprolide (agonist) and cetrorelix (antagonist) on cell growth, apoptosis and GnRH receptor expression using a primary cell coculture system. Also, binding characteristics of prostate GnRH receptor in this culture system are described. Epithelial and stromal cells were obtained from prostate adenocarcinoma samples and cocultured in a bicameral system. Expression of GnRH receptors was evaluated by semiquantitative RT-PCR (transcript level) and Western blot (protein level). Cell growth was estimated by MTT method and apoptosis by DNA fragmentation using COMET assay. Saturation and competition binding studies were carried out using 125I-GnRH as radioligand. GnRH receptors from cell cultures of prostate cancer exhibited a single class of binding sites with a Kd of 1.11 +/- 0.28 nM and a Bmax of 2.81 +/- 0.37 pmol/mg of membrane protein for GnRH. Leuprolide and cetrorelix showed no effect on GnRH receptor expression. Both analogues showed a significant reduction in cell growth rate and an increase in DNA-fragmented cell number. These effects were dependent on the analogue concentrations (from 5-20 ng/mL). Considering that the culture system used in this work represents more closely the in vivo conditions of tumor cells than metastatic derived cell lines, we conclude that GnRH analogues have a significant inhibitory effect on cell viability of cells expressing GnRH receptors. In addition, GnRH receptors expressed in tumor prostatic cells seem not discriminate between agonist and antagonist, both analogues activating these receptors. Also, leuprolide and cetrorelix treatments did not influence GnRH receptor expression in our culture system. These differences with pituitary receptors may be explained by differences in affinity, transduction mechanism and molecular context in prostatic tissue.

Role of the phosphatase PP4 in the activation of JNK-1 in prostate carcinoma cell lines PC-3 and LNCaP resulting in increased AP-1 and EGR-1 activity.

The specific signaling connections between the mitogen-activated protein kinases (MAPK) such as c-Jun N-terminal kinase (JNK-1) and phosphatases PP4 and M3/6, affecting the family of early nuclear factors, is complex and remains poorly understood. JNK-1 regulates cellular differentiation, apoptosis and stress responsiveness by up-regulating early nuclear factors such as c-Jun, a member of the activating protein (AP-1) family, and the Early Growth Factor (EGR-1). C-Jun, when phosphorylated by c-Jun N-terminal kinase (JNK-1) associates with c-Fos to form the AP-1 transcription factor that activates gene expression. We have investigated the regulation of the JNK-1 kinase by co-transfecting phosphatases PP4 and M3/6 in prostate cancer cell lines PC-3 and LNCaP, which have been previously stimulated with human EGF or cisplatin. Co-transfections of plasmids expressing the JNK-1 and the serine/threonine phosphatases PP4 resulted in a significant increase in JNK-1 activity in both PC3 and LNCaP cells. In contrast, co-transfection of JNK-1 with the dual specific phosphatase serine/threonine M3/6 showed only a marginal effect in JNK-1 activity. The phosphatase M3/6 also failed in blocking the induction of JNK-1 activity observed in presence of PP4. The higher activity of JNK-1 was associated with increased activities of the factors c-Jun/AP-1 and EGR-1. This suggests that JNK-1 activity in PC-3 and LNCaP cells requires not only active PP4 for stable maintenance but also suggests that the relative degree of phosphorylation of multiple cellular components is the determinant of JNK-1 stability.

Secretion of prostatic specific antigen, proliferative activity and androgen response in epithelial-stromal co-cultures from human prostate carcinoma.

We investigate the proliferative activity, prostatic specific antigen (PSA) secretion, morphology and androgen response of human prostate tumour epithelial cells co-cultured with stromal cells in a bicameral system. Stromal and epithelial cells were isolated from prostate adenocarcinoma by enzyme digestion and cultured in defined media. Immunocytochemistry for prostate carcinoma tumour antigen (PCTA-1) was performed for culture purity evaluation. Also, the morphology of the epithelial cells in co-culture was evaluated by electron microscopy. PSA was determined by microparticle enzyme immunoassay (MEIA) automatized protocol and the proliferation was evaluated by a commercial spectrophotometric kit, based on formazan salt formation. Both cell cultures showed more than 90% of purity. The epithelial cell co-cultures showed marked membrane processes and cell interdigitations. The proliferative activity of the epithelial cells was increased in presence of stromal cells. Also, PSA secretion was significantly increased and maintained for at least 14 days, whereas the androgen response for PSA secretion was evidenced only in co-culture condition. Primary co-cultures of epithelial and stromal cells from human prostate carcinoma are able to maintain, for a prolonged time, proliferative and secretory properties as well hormone response, and represent a valuable tool for cellular and molecular studies on prostate cancer.