Tick saliva inhibits the chemotactic function of MIP-1 alpha and selectively impairs chemotaxis of immature dendritic cells by down-regulating cell-surface CCR5

Ticks are blood-feeding arthropods that secrete immunomodulatory molecules through their saliva to antagonize host inflammatory and immune responses. As dendritic cells (DCs) play a major role in host immune responses, we studied the effects of Rhipicephalus sanguineus tick saliva on DC migration and function. Bone marrow-derived immature DCs pre-exposed to tick saliva showed reduced migration towards macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta and regulated upon activation, normal T cell expressed and secreted (RANTES) chemokines in a Boyden microchamber assay. This inhibition was mediated by saliva which significantly reduced the percentage and the average cell-surface expression of CC chemokine receptor CCR5. In contrast, saliva did not alter migration of DCs towards MIP-3 beta, not even if the cells were induced for maturation. Next, we evaluated the effect of tick saliva on the activity of chemokines related to DC migration and showed that tick saliva per se inhibits the chemotactic function of MIP-1 alpha, while it did not affect RANTES, MIP-1 beta and MIP-3 beta. These data suggest that saliva possibly reduces immature DC migration, while mature DC chemotaxis remains unaffected. In support of this, we have analyzed the percentage of DCs on mice 48 h after intradermal inoculation with saliva and found that the DC turnover in the skin was reduced compared with controls. Finally, to test the biological activity of the saliva-exposed DCs, we transferred DCs pre-cultured with saliva and loaded with the keyhole limpet haemocyanin (KLH) antigen to mice and measured their capacity to induce specific T cell cytokines. Data showed that saliva reduced the synthesis of both T helper (Th)1 and Th2 cytokines, suggesting the induction of a non-polarised T cell response. These findings propose that the inhibition of DCs migratory ability and function may be a relevant mechanism used by ticks to subvert the immune response of the host. (c) 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Tick saliva induces regulatory dendritic cells: MAP-kinases and Toll-like receptor-2 expression as potential targets

Ticks (Acari: Ixodidae) are bloodsucking ectoparasitic arthropods of human and veterinary medical importance. Tick saliva has been shown to contain a wide range of bioactive molecules with vasodilatory, antihemostatic, and immunomodulatory activities. We have previously demonstrated that saliva from Rhipicephalus sanguineus ticks inhibits the maturation of dendritic cells (DCs) stimulated with LPS. Here we examined the mechanism of this immune subversion, evaluating the effect of tick saliva on Toll-like receptor (TLR)-4 signalling pathway in bone marrow-derived DCs. We demonstrated that R. sanguineus tick saliva impairs maturation of DCs stimulated with LIPS, a TLR-4 ligand, leading to increased production of interleukin (IL)-10 and reduced synthesis of IL-12p70 and TNF-alpha. The immunomodulatory effect of the tick saliva on the production of pro-inflammatory cytokines by DCs stimulated with LPS was associated with the observation that tick saliva inhibits the activation of the ERK 1/2 and p38 MAP kinases. These effects were independent of the expression of TLR-4 on the surface of DCs. Additionally, saliva-treated DCs also presented a similar pattern of cytokine modulation in response to other TLR ligands. Since the recent literature reports that several parasites evade immune responses through TLR-2-mediated production of IL-10, we evaluated the effect of tick saliva on the percentage of TLR-2(+) DCs stimulated with the TLR-2 ligand lipoteicoic acid (LTA). The data showed that the population of DCs expressing TLR-2 was significantly increased in DCs treated with LTA plus saliva. In addition, tick saliva alone increased the expression of TLR-2 in a dose- and time-dependent manner. Our data suggest that tick saliva induces regulatory DCs, which secrete IL-10 and low levels of IL-12 and TNF-alpha when stimulated by TLR ligands. Such regulatory DCs are associated with expression of TLR-2 and inhibition of ERK and p38, which promotes the production of IL-10 and thus down-modulates the host`s immune response, possibly favouring susceptibility to tick infestations. (C) 2009 Elsevier B.V. All rights reserved.

Study of lymphocyte subpopulations in bone marrow in a model of protein-energy malnutrition

Objective: Protein-energy malnutrition (PEM) is an important public health problem affecting millions of people worldwide. Hematopoietic tissue requires a high nutrient supply, and a reduction in leukocytes, especially lymphocytes, suggests that some nutritional deficiencies might be altering bone marrow function and decreasing its ability to produce lymphocytes. In this study, we evaluated the effect that PEM has on lymphocyte subtypes and the cell cycle of CD5(+) cells. Methods: Swiss mice were subjected to PEM using a low-protein diet containing 4% protein. When the experimental group had lost about 20% of their original body weight, we collected blood and bone marrow cells and evaluated the hemogram, the myelogram, bone marrow lymphoid markers using flow cytometry, and the cell cycle in CD5(+) bone marrow. Results: Malnourished animals presented anemia, reticulocytopenia, and leukopenia with lymphopenia. The bone marrow was hypocellular, and flow cytometric analyses of bone marrow cells showed cells that were CD45(+) (91.2%), CD2(+) (84.9%), CD5(+) (37.3%), CD3(+) (23.5%), CD19(+) (43.3%), CD22(+) (34.7%), CD19(+)/CD2(+) (51.2%), CD19(+)/CD3(+)(24.0%), CD19(+)/CD5(+) (13.2%), CD22(+)/CD2(+) (40.1%), CD22(+)/CD3(+) (30.3%), and CD22(+)/CD5(+) (1.1%) in malnourished animals and CD45(+) (97.5%), CD2(+) (42.9%), CD5(+) (91.5%), CD3(+) (92.0%), CD19(+) (52.0%), CD22(+) (75.6%), CD19(+)/CD2(+) (62.0%), CD19(+)/CD3(+) (55.4%), CD19(+)/CO5(+) (6.7%), CD22(+)/CD2(+) (70.3%), CD22(+)/CD3(+) (55.9%), and CD22(+)/ CD5(+) (8.4%) in control animals. Malnourished animals also presented more CD5(+) cells in the G0 phase of cell cycle development. Conclusion: Malnourished animals presented bone marrow hypoplasia, maturation interruption, prominent lymphopenia with depletion in the lymphoid lineage, and changes in cellular development. We suggest that these changes are some of the primary causes of lymphopenia in cases of PEM and partly explain the increase in susceptibility to infections found in malnourished individuals. Published by Elsevier Inc.

Population balance model of in vivo neutrophil formation following bone marrow rescue therapy

In this paper, we develop a simple four parameter population balance model of in vivo neutrophil formation following bone marrow rescue therapy. The model is used to predict the number and type of neutrophil progenitors required to abrogate the period of severe neutropenia that normally follows a bone marrow transplant. The estimated total number of 5 billion neutrophil progenitors is consistent with the value extrapolated from a human trial. The model provides a basis for designing ex vivo expansion protocols.

Immunobiology of liver dendritic cells

Dendritic cells (DC) are rare, bone marrow-derived antigen-presenting cells that play a critical role in the induction and regulation of immune reactivity. In this article, we review the identification and characterization of liver DC, their ontogenic development, in vivo mobilization and population dynamics. In addition, we discuss the functions of DC isolated from liver tissue or celiac lymph, or propagated in vitro from liver-resident haemopoietic stem/progenitor cells. Evidence concerning the role of DC in viral hepatitis. liver tumours, autoimmune liver diseases, granulomatous inflammation and the outcome of liver transplantation is also discussed.

Persistence of recipient lymphocytes in NOD mice after irradiation and bone marrow transplantation

The non-obese diabetic (NOD) mouse is a unique and invaluable model of autoimmune disease, in particular type I diabetes. Bone marrow transplantation as a therapy for type I diabetes has been explored in NOD mice. NOD mice require higher doses of conditioning irradiation for successful allogeneic bone marrow transplantation, suggesting that NOD hematopoietic cells are radioresistant compared to those of other mouse strains. However, studies of hematopoietic reconstitution in NOD mice are hampered by the lack of mice bearing a suitable cell-surface marker that would allow transferred cells or their progeny to be distinguished. In order to monitor hematopoietic reconstitution in NOD mice we generated congenic NOD mice that carry the alternative allelic form of the pan-leukocyte alloantigen CD45. Following irradiation and congenic bone marrow transplantation, we found that the myeloid lineage was rapidly reconstituted by cells of donor origin but substantial numbers of recipient T lymphocytes persisted even after supra-lethal irradiation. This indicates that radiation resistance in the NOD hematopoietic compartment is a property primarily of mature T lymphocytes. (C) 2004 Elsevier Ltd. All rights reserved.

Serum Ferritin Level Remains a Reliable Marker of Bone Marrow Iron Stores Evaluated by Histomorphometry in Hemodialysis Patients

Background and objectives: As well as being a marker of body iron stores, serum ferritin (sFerritin) has also been shown to be a marker of inflammation in hemodialysis (HD) patients. The aim of this study was to analyze whether sFerritin is a reliable marker of the iron stores present in bone marrow of HD patients. Design: Histomorphometric analysis of stored transiliac bone biopsies was used to assess iron stores by determining the number of iron-stained cells per square millimeter of bone marrow. Results: In 96 patients, the laboratory parameters were hemoglobin = 11.3 +/- 1.6 g/dl, hematocrit = 34.3 +/- 5%, sFerritin 609 +/- 305 ng/ml, transferrin saturation = 32.7 +/- 22.5%, and C-reactive protein (CRP) = 0.9 +/- 1.4 mg/dl. sFerritin correlated significantly with CRP, bone marrow iron, and time on HD treatment W = 0.006, 0.001, and 0.048, respectively). The independent determinants of sFerritin were CRP (beta-coef = 0.26; 95% CI = 24.6 to 132.3) and bone marrow iron (beta-coef = 0.32; 95% CI = 0.54 to 2.09). Bone marrow iron was higher in patients with sFerritin >500 ng/ml than in those with sFerritin :5500 ng/ml. In the group of patients with sFerritin :5500 ng/ml, the independent determinant of sFerritin was bone marrow iron (beta-coef = 0.48, 95% CI = 0.48 to 1.78), but in the group of patients with sFerritin >500 ng/ml, no independent determinant of sFerritin was found. Conclusions: sFerritin adequately reflects iron stores in bone marrow of HD patients.

NFAT1 transcription factor is central in the regulation of tissue microenvironment for tumor metastasis

Members of the nuclear factor of activated T cell (NFAT) family of transcription factors were originally described in T lymphocytes but later shown to be expressed in several immune and non-immune cell types. NFAT proteins can modulate cellular transformation intrinsically, and NFAT-deficient (NFAT1-/-) mice are indeed more susceptible to transformation than wild-type counterparts. However, the contribution of an NFAT1-/- microenvironment to tumor progression has not been studied. We have addressed this question by inoculating NFAT1-/- mice with B16F10 melanoma cells intravenously, an established model of tumor homing and growth. Surprisingly, NFAT1-/- animals sustained less tumor growth in the lungs after melanoma inoculation than wild-type counterparts. Even though melanoma cells equally colonize NFAT1-/- and wild-type lungs, tumors do not progress in the absence of NFAT1 expression. A massive mononuclear perivascular infiltrate and reduced expression of TGF-beta in the absence of NFAT1 suggested a role for tumor-infiltrating immune cells and the cytokine milieu. However, these processes are independent of an IL-4-induced regulatory tumor microenvironment, since lack of this cytokine does not alter the phenotype in NFAT1-/- animals. Bone marrow chimera experiments meant to differentiate the contributions of stromal and infiltrating cells to tumor progression demonstrated that NFAT1-induced susceptibility to pulmonary tumor growth depends on NFAT1-expressing parenchyma rather than on bone marrow-derived cells. These results suggest an important role for NFAT1 in radio-resistant tumor-associated parenchyma, which is independent of the anti-tumor immune response and Th1 versus Th2 cytokine milieu established by the cancer cells, but able to promote site-specific tumor growth.

EVALUATION OF CENTRIFUGED OSTEOGENIC BONE MARROW IN FRACTURE CONSOLIDATION IN RABBITS

Objective The purpose of this study was to evaluate the efficacy of a centrifuged osteogenic bone marrow aspirate to stimulate healing in rabbit fibular osteotomies Methods Ten white New Zealand rabbits were used A transverse medial diaphyseal fibular osteotomy was performed on the right fibula where an absorbable collagen sponge embedded in osteogenic centrifuged bone marrow aspirate obtained from the ipsilateral iliac bone was inserted The left fibula was used as the control group where the collagen absorbable sponge was inserted without the osteogenic centrifuged aspirate The rabbits were sacrificed four weeks after surgery to evaluate bone callus formation Analyses of results were performed with DEXA bone densitometry to evaluate callus mineral mass multislice computed tomography to evaluate callus volume and histomorphometry to evaluate the relative rate of tissue formation Results The employment of centrifuged osteogenic bone marrow aspirate resulted in a 40 3% increase of callus bone mineral mass and increased relative quantity of bone tissue formation by 9 4% without a significant increase in the relative quantities of cartilage fibrous tissue or in callus volume Conclusions This study shows that the centrifuged osteogenic bone marrow aspirate was able to improve the healing of experimental fibular osteotomies in rabbits

Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and gene-expression profile with CD146(+) perivascular cells and fibroblasts

Objective. The relationship of multipotent mesenchymal stromal cells (MSC) with pericytes and fibroblasts has not been established thus far, although they share many markers of primitive marrow stromal cells and the osteogenic, adipogenic, and chondrogenic differentiation potentials. Materials and Methods. We compared MSCs from adult or fetal tissues, MSC differentiated in vitro, fibroblasts and cultures of retinal pericytes obtained either by separation with anti-CD146 or adhesion. The characterizations included morphological, immunophenotypic, gene-expression profile, and differentiation potential. Results. Osteogenic, adipocytic, and chondrocytic differentiation was demonstrated for MSC, retinal perivascular cells, and fibroblasts. Cell morphology and the phenotypes defined by 22 markers were very similar. Analysis of the global gene expression obtained by serial analysis of gene expression for 17 libraries and by reverse transcription polymerase chain reaction of 39 selected genes from 31 different cell cultures, revealed similarities among MSC, retinal perivascular cells, and hepatic stellate cells. Despite this overall similarity, there was a heterogeneous expression of genes related to angiogenesis, in MSC derived from veins, artery, perivascular cells, and fibroblasts. Evaluation of typical pericyte and MSC transcripts, such as NG2, CD146, CD271, and CD140B on CD146 selected perivascular cells and MSC by real-time polymerase chain reaction confirm the relationship between these two cell types. Furthermore, the inverse correlation between fibroblast-specific protein-1 and CD146 transcripts observed on pericytes, MSC, and fibroblasts highlight their potential use as markers of this differentiation pathway. Conclusion. Our results indicate that human MSC and pericytes are similar cells located in the wall of the vasculature, where they function as cell sources for repair and tissue maintenance, whereas fibroblasts are more differentiated cells with more restricted differentiation potential. (C) 2008 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Cysteinyl-leukotriene type 1 receptors transduce a critical signal for the up-regulation of eosinophilopoiesis by interleukin-13 and eotaxin in murine bone marrow

IL-13 and eotaxin play important, inter-related roles in asthma models. In the lungs, CysLT, produced by the 5-LO-LTC4S pathway, mediate some local responses to IL-13 and eotaxin; in bone marrow, CysLT enhance IL-5-dependent eosinophil differentiation. We examined the effects of IL-13 and eotaxin on eosinophil differentiation. Semi-solid or liquid cultures were established from murine bone marrow with GM-CSF or IL-5, respectively, and the effects of IL-13, eotaxin, or CysLT on eosinophil colony formation and on eosinophil differentiation in liquid culture were evaluated, in the absence or presence of: a) the 5-LO inhibitor zileuton, the FLAP inhibitor MK886, or the CysLT1R antagonists, montelukast and MK571; b) mutations that inactivate 5-LO, LTC4S, or CysLT1R; and c) neutralizing mAb against eotaxin and its CCR3 receptor. Both cytokines enhanced GM-CSF-dependent eosinophil colony formation and IL-5-stimulated eosinophil differentiation. Although IL-13 did not induce eotaxin production, its effects were abolished by anti-eotaxin and anti-CCR3 antibodies, suggesting up-regulation by IL-13 of responses to endogenous eotaxin. Anti-CCR3 blocked eotaxin completely. The effects of both cytokines were prevented by zileuton, MK886, montelukast, and MK571, as well as by inactivation of the genes coding for 5-LO, LTC4S, and CysLT1R. In the absence of either cytokine, these treatments or mutations had no effect. These findings provide evidence for: a) a novel role of eotaxin and IL-13 in regulating eosinophilopoiesis; and b) a role for CysLTRs in bone marrow cells in transducing cytokine regulatory signals. J. Leukoc. Biol. 87: 885-893; 2010.

Characterization of Equine Adipose Tissue-Derived Progenitor Cells Before and After Cryopreservation

In horses, stem cell therapies are a promising tool to the treatment of many injuries, which are common consequences of athletic endeavor, resulting in high morbidity and often compromising the performance. In spite of many advantages, the isolation of stem cells similar to human, from equine adipose tissue, occurred only recently. The aim of this study was to isolate equine adipose tissue-derived progenitor cells (eAT-PC), to characterize their proliferative potential, and to study their differentiation capacity before and after cryopreservation. The cells, isolated from horse adipose tissue, presented similar fibroblast-like cell morphology in vitro. Their proliferation rate was evaluated during 63 days (23 passages) before and after cryopreservation. After the induction of osteogenic differentiation, von Kossa staining and positive immunostaining studies revealed the formation of calcified extracellular matrix confirming the osteogenic potential of these cells. Adipogenic differentiation was induced using two protocols: routine and other one developed by us, while our protocol requires a shorter time (Oil Red O staining revealed significant accumulation of lipid droplets after 7 days). Chondrogenic differentiation was observed after 21 days of induced pellet culture, as evidenced by histological (toluidine blue) and immunohistochemistry studies. Our data demonstrate that eAT-PC can be easily isolated and successfully expanded in vitro while presenting significant proliferating rate. These cells can be maintained undifferentiated in vitro and can efficiently undergo differentiation at least into mesodermal derivates. These eAT-PC properties were preserved even after cryopreservation. Our findings classify eAT-PC as a promising type of progenitor cells that can be applied in different cell therapies in equines.

Low doses of monocrotaline in rats cause diminished bone marrow cellularity and compromised nitric oxide production by peritoneal macrophages

Monocrotaline (MCT) is a pyrrolizidine alkaloid found in a variety of plants. The main symptoms of MCT toxicosis in livestock are related to hepato- and nephrotoxicity; in rodents and humans, the induction of a pulmonary hypertensive state that progresses to cor pulmonale has received much attention. Although studies have shown that MCT can cause effects on cellular functions that would be critical to those of lymphocytes/macrophages during a normal immune response, no immunotoxicological study on MCT have yet to ever be performed. Thus, the aim of the present study was to evaluate the effect of MCT on different branches of the immune system using the rat - which is known to be sensitive to the effects of MCT - as the model. Rats were treated once a day by gavage with 0.0, 0.3, 1.0, 3.0, or 5.0 mg MCT/kg for 14 days, and then any effects of the alkaloid on lymphoid organs, acquired immune responses, and macrophage activity were evaluated. No alterations in the relative weight of lymphoid organs were observed; however, diminished bone marrow cellularity in rats treated with the alkaloid was observed. MCT did not affect humoral or cellular immune responses. When macrophages were evaluated, treatments with MCT caused no significant alterations in phagocytic function or in hydrogen peroxide (H(2)O(2)) production; however, the MCT did cause compromised nitric oxide (NO) release by these cells.

Unravelling the role of alpha 2,6 sialic acid on mouse dentritic cells functions

RESUMO: As células dendríticas (CDs) são fundamentais na imunomodulação e iniciação de respostas imunes adaptativas, enquanto os ácidos siálicos (Sias) são potenciais imunomoduladores. Estas células expressam níveis elevados da sialiltransferase ST6Gal-1, que transfere Sias para a posição terminal de oligossacáridos. De facto, a maturação de CDs está associada a uma diminuição da sialilação na sua superfície celular. Apesar de ter função biológica desconhecida, a forma solúvel, extracelular de ST6Gal-1 aumenta em cancros e inflamação. Ainda assim, esta foi recentemente identificada como moduladora da hematopoiese. Considerando o importante papel das CDs na iniciação de respostas anticancerígenas, uma ligação entre a sialilação extrínseca induzida por ST6Gal-1 extracelular e o seu papel na modulação de CDs deve ser identificada. Neste trabalho hipotetizou-se que a sialilação α2,6 extrínseca de CDs diminui o seu perfil de maturação mediante ativação por lipopolissacarídeo (LPS). O objetivo principal foi sialilar extrinsecamente em α2,6 CDs da medula óssea de murganhos, avaliando os seus perfis de maturação e de libertação de citocinas, após estimulação com LPS (por Citometria de Fluxo e ELISA, respetivamente). Ao contrário da hipótese, o perfil celular não foi modulado, usando várias abordagens. Por outro lado, a consequência da falta de α2,6 Sias na maturação de CDs foi avaliada analisando: 1) CDs da medula óssea de murganhos tratadas com sialidase, 2) CDs da medula óssea e 3) CDs das vias aéreas, ambas de murganhos deficientes em ST6Gal-1, comparando com a estirpe selvagem. Estes resultados sugerem que a perta total de α2,6 Sias se relaciona com o aumento da expressão do complexo de histocompatibilidade principal de classe II. Apesar de controverso, é provável existirem mecanismos inerentes à ativação por LPS, reduzindo a eficácia de ST6Gal-1 extracelular. Por outro lado, a modificação no perfil de CDs de murganhos deficientes em ST6Gal-1 poderá relacionar-se com uma predisposição para um estado inflamatório severo. Com isto, o trabalho desenvolvido abriu futuras linhas de investigação, nomeadamente explorar outros fatores envolvidos na (de)sialilação α2,6 de CDs, podendo ter impacto em imunoterapia com uso de CDs.--------------------------ABSTRACT: Dendritic cells (DCs) are vital for immunomodulation and the initiation of adaptive immune responses, whereas sialic acids (Sias) are potential immunomodulators. These cells express high levels of sialyltransferase ST6Gal-1, responsible for transferring Sias to the terminal position of oligosaccharide chains. Indeed, DCs’ maturation is associated with decreased cell surface sialylation. Although its biological significance is unknown, the soluble, extracellular form of ST6Gal-1 increases in cancers and inflammation. However, extracellular ST6Gal-1 was recently identified as modulator of hematopoiesis. Considering that DCs play a crucial role in the initiation of a productive anti-cancer immune response, a link between extrinsic sialylation by the extracellular ST6Gal-1 on DC function needs to be investigated. We hypothesize that extrinsic α2,6 sialylation of DCs diminishes their maturation features upon lipopolysaccharide (LPS) stimulation. The main goal was to extrinsically α2,6 sialylate mice bone marrow derived DCs (BMDCs) and to evaluate their maturation and cytokine profiles upon LPS stimulation (by Flow Cytometry and ELISA, respectively). Unlike the hypothesis, we observed that BMDCs’ profile is not modulated, even using several approaches. In contrast, the consequence of lacking cell surface α2,6 Sias in DC maturation was assessed by analysing: 1) sialidase treated BMDCs, 2) BMDCs from mice lacking ST6Gal-1 and 3) DCs from mice airways, comparing wild type with ST6Gal-1 knockout mice. These results suggest that overall lack in α2,6 Sias is related with increased expression of major histocompatibility class II (MHC-II). Although appearing to be controversial findings, other intracellular mechanisms might be occurring upon LPS-induced BMDC activation, probably reducing extracellular ST6Gal-1 effect. In opposite, the modification observed in DC profile of ST6Gal-1 knockout mice might be related to its predisposition to a more severe inflammatory status. With this, the developed work opened future lines of investigation, namely exploring other factors involved in α2,6 (de)sialylation of DC, which might have influence in immunotherapy using DCs.

A novel gene expression signature in peripheral blood mononuclear cells for early detection of colorectal cancer.

BACKGROUND: Early detection and treatment of colorectal adenomatous polyps (AP) and colorectal cancer (CRC) is associated with decreased mortality for CRC. However, accurate, non-invasive and compliant tests to screen for AP and early stages of CRC are not yet available. A blood-based screening test is highly attractive due to limited invasiveness and high acceptance rate among patients. AIM: To demonstrate whether gene expression signatures in the peripheral blood mononuclear cells (PBMC) were able to detect the presence of AP and early stages CRC. METHODS: A total of 85 PBMC samples derived from colonoscopy-verified subjects without lesion (controls) (n = 41), with AP (n = 21) or with CRC (n = 23) were used as training sets. A 42-gene panel for CRC and AP discrimination, including genes identified by Digital Gene Expression-tag profiling of PBMC, and genes previously characterised and reported in the literature, was validated on the training set by qPCR. Logistic regression analysis followed by bootstrap validation determined CRC- and AP-specific classifiers, which discriminate patients with CRC and AP from controls. RESULTS: The CRC and AP classifiers were able to detect CRC with a sensitivity of 78% and AP with a sensitivity of 46% respectively. Both classifiers had a specificity of 92% with very low false-positive detection when applied on subjects with inflammatory bowel disease (n = 23) or tumours other than CRC (n = 14). CONCLUSION: This pilot study demonstrates the potential of developing a minimally invasive, accurate test to screen patients at average risk for colorectal cancer, based on gene expression analysis of peripheral blood mononuclear cells obtained from a simple blood sample.