TRIM28 and ß-actin identified via nanobody-based reverse proteomics approach as possible human glioblastoma biomarkers.

Malignant gliomas are among the rarest brain tumours, and they have the worst prognosis. Grade IV astrocytoma, known as glioblastoma multiforme (GBM), is a highly lethal disease where the standard therapies of surgery, followed by radiation and chemotherapy, cannot significantly prolong the life expectancy of the patients. Tumour recurrence shows more aggressive form compared to the primary tumour, and results in patient survival from 12 to 15 months only. Although still controversial, the cancer stem cell hypothesis postulates that cancer stem cells are responsible for early relapse of the disease after surgical intervention due to their high resistance to therapy. Alternative strategies for GBM therapy are thus urgently needed. Nanobodies are single-domain antigen-binding fragments of heavy-chain antibodies, and together with classical antibodies, they are part of the camelid immune system. Nanobodies are small and stable, and they share a high degree of sequence identity to the human heavy chain variable domain, and these characteristics offer them advantages over classical antibodies or antibody fragments. We first immunised an alpaca with a human GBM stem-like cell line prepared from primary GBM cultures. Next, a nanobody library was constructed in a phage-display vector. Using nanobody phage-display technology, we selected specific GBM stem-like cell binders through a number of affinity selections, using whole cell protein extracts and membrane protein-enriched extracts from eight different GBM patients, and membrane protein-enriched extracts from two established GBM stem-like cell lines (NCH644 and NCH421K cells). After the enrichment, periplasmic extract ELISA was used to screen for specific clones. These nanobody clones were recloned into the pHEN6 vector, expressed in Escherichia coli WK6, and purified using immobilised metal affinity chromatography and size-exclusion chromatography. Specific nanobody:antigen pairs were obtained and mass spectrometry analysis revealed two proteins, TRIM28 and ß-actin, that were up-regulated in the GBM stem-like cells compared to the controls.

Single-domain antibodies as building blocks for novel therapeutics.

Antibodies are large and complex molecules, with two identical parts that bind independently of each other onto the antigen and the third part of the molecule that dictates the effector function(s). To improve the therapeutic value of antibodies, protein-engineering endeavors reduced the size of the antigen-binding moiety to a single-domain unit. Occasionally, it was demonstrated that the single-domain antigen-binding derivatives of antibodies can have--on their own--an agonistic (or antagonistic) effect on their target. The small size and strict monomeric behavior, in combination with other biochemical properties such as high solubility and high specificity and affinity for the cognate antigen, make single-domain antibodies ideal to design novel man-made conjugates harnessed with innovative effector functions outside the reach of classical antibodies.

Antibody technology in proteomics.

Today's proteomic analyses are generating increasing numbers of biomarkers, making it essential to possess highly specific probes able to recognize those targets. Antibodies are considered to be the first choice as molecular recognition units due to their target specificity and affinity, which make them excellent probes in proteomics. In the post-genomic era and with high-throughput techniques available, the goal is to discriminate between all individual proteins from the proteome including their splice variants and post-translationally modified derivatives. Aided by advances in generation, selection and engineering of antibody-based recognition units, antibody fragments provide tools for detection of high- as well as low-abundant analytes even in complex, non-fractionated proteomes in conjunction with usage of small amounts of samples and reagents. In addition, large consortia aim at generating vast numbers of antibody-based recognition units suitable for future diagnostics and therapeutics.

Alternatively activated myeloid cells limit pathogenicity associated with African trypanosomiasis through the IL-10 inducible gene selenoprotein P.

Uncontrolled inflammation is a major cause of tissue injury/pathogenicity often resulting in death of a host infected with African trypanosomes. Thus, comparing the immune response in hosts that develop different degrees of disease severity represents a promising approach to discover processes contributing to trypanosomiasis control. It is known that limitation of pathogenicity requires a transition in the course of infection, from an IFN-gamma-dependent response resulting in the development of classically activated myeloid cells (M1), to a counterbalancing IL-10-dependent response associated with alternatively activated myeloid cells (M2). Herein, mechanisms and downstream effectors by which M2 contribute to lower the pathogenicity and the associated susceptibility to African trypanosomiasis have been explored. Gene expression analysis in IL-10 knockout and wild-type mice, that are susceptible and relatively resistant to Trypanosoma congolense infection, respectively, revealed a number of IL-10-inducible genes expressed by M2, including Sepp1 coding for selenoprotein P. Functional analyses confirm that selenoprotein P contributes to limit disease severity through anti-oxidant activity. Indeed, Sepp1 knockout mice, but not Sepp1(Delta)(240-361) mice retaining the anti-oxidant motif but lacking the selenium transporter domain of selenoprotein P, exhibited increased tissue injury that associated with increased production of reactive oxygen species and increased apoptosis in the liver immune cells, reduced parasite clearance capacity of myeloid cells, and decreased survival. These data validate M2-associated molecules as functioning in reducing the impact of parasite infection on the host.

The metastatic T-cell hybridoma antigen/P-selectin glycoprotein ligand 1 is required for hematogenous metastasis of lymphomas.

Using variants of the murine BW5147 lymphoma cell-line, we have previously identified 3 monoclonal antibodies (MAbs) that discriminate between metastatic and nonmetastatic BW5147-derived T-cell hybridomas and lymphomas, as well as BW5147-unrelated T-lymphomas. These MAbs were reported to recognize an identical membrane-associated sialoglycoprotein, termed "metastatic T-cell hybridoma antigen" (MTH-Ag). Here, we document that the expression pattern of the MTH-Ag on metastatic and nonmetastatic BW5147 variants correlates with that of the P-selectin glycoprotein ligand 1 (PSGL-1), a sialomucin involved in leukocyte recruitment to sites of inflammation. Moreover, the MAbs against the MTH-Ag recognize PSGL-1 when it is transfected in MTH-Ag-negative BW5147 variants, suggesting that the MTH-Ag is PSGL-1. Overexpression of MTH-Ag/PSGL-1 in MTH-Ag-negative BW5147 variants did not affect their in vivo malignancy. Yet, down-regulation of MTH-Ag/PSGL-1 expression on metastatic, MTH-Ag-positive BW5147 variants, using an RNA interference (RNAi) approach, resulted, in a dose-dependent manner, in a significant reduction of liver and spleen colonization and a delay in mortality of the recipient mice upon intravenous inoculation. Collectively, these results demonstrate that, although MTH-Ag/PSGL-1 overexpression alone may not be sufficient for successful dissemination and organ colonization, MTH-Ag/PSGL-1 plays a critical role in hematogenous metastasis of lymphoid cancer cells.

Alternatively activated macrophages in protozoan infections.

A type 1 cytokine-dependent pro-inflammatory response inducing classically activated macrophages is crucial for parasite control during protozoan infections but can also contribute to the development of immunopathological disease symptoms. Accumulating evidence indicates that interleukins 4, 13 and 10, transforming growth factor-beta, immune complexes and apoptotic cells elicited during these infections induce alternative activation states of macrophages, affecting disease outcome by, on the one hand, promoting parasite survival and proliferation and, on the other hand, limiting collateral tissue damage because of excessive type 1 inflammation. Thus, modulation of macrophage activation may be instrumental in allowing parasite persistence and long-term host survival.

The tumor-promoting effect of TNF-alpha involves the induction of secretory leukocyte protease inhibitor.

According to the cancer immunoediting concept, inflammatory mediators play not only a critical role in promoting host protection against cancer but also contribute to cancer cell growth and survival. TNF-alpha is a critical factor in this network. However, the mechanisms underlying the tumor-promoting effect of TNF-alpha have not been fully elucidated yet. We previously reported that in vitro culture of Lewis lung carcinoma 3LL cells with TNF-alpha-producing macrophages resulted in enhanced resistance toward TNF-alpha-mediated lysis and increased malignancy of the 3LL cells. In this study, we analyzed the effects of endogenous TNF-alpha on TNF-alpha resistance and malignant behavior in vivo of low-malignant/TNF-alpha-sensitive 3LL-S cells and cancer cells derived from 3LL-S tumors that developed in wild-type or TNF-alpha(-/-) mice. Interestingly, 3LL-S cells acquired a malignant phenotype in vivo depending on the presence of host TNF-alpha, whereas acquisition of TNF-alpha resistance was TNF-alpha-independent. This result suggested that malignancy-promoting characteristics of 3LL-S cells other than TNF-alpha resistance are influenced in vivo by TNF-alpha. We previously identified the malignancy-promoting genes, secretory leukocyte protease inhibitor (SLPI) and S100A4, as being up-regulated in 3LL-S cells upon their s.c. growth in wild-type mice. In this study, we show that SLPI, but not S100A4, was induced in 3LL-S cells both in vitro and in vivo by TNF-alpha, and that silencing of in vivo induced 3LL-S SLPI expression using RNA interference abrogated in vivo progression but did not influence TNF-alpha resistance. These data indicate that SLPI induction may be one mechanism whereby TNF-alpha acts as an endogenous tumor promoter.

Identification of a common gene signature for type II cytokine-associated myeloid cells elicited in vivo in different pathologic conditions.

Compared with type I cytokine-associated myeloid (M1) cells, the molecular repertoire and mechanisms underlying functional properties of type II cytokine-associated myeloid (M2) cells are poorly characterized. Moreover, most studies have been limited to in vitro-elicited M2 cells. Here, comparative gene expression profiling of M1 and M2 cells, elicited in murine models of parasitic infections and cancer, yielded a common signature for in vivo-induced M2 populations independent of disease model, mouse strain, and organ source of cells. Some of these genes, such as cadherin-1, selenoprotein P, platelet-activating factor acetylhydrolase, and prosaposin, had not been documented as associated with M2. Overall, the common signature genes provide a molecular basis for a number of documented or suggested properties of M2, including immunomodulation, down-regulation of inflammation, protection against oxidative damage, high capacity for phagocytosis, and tissue repair. Interestingly, several common M2 signature genes encode membrane-associated markers that could be useful for the identification and isolation of M2. Some of these genes were not induced by IL-4/IL-13 or IL-10 under various in vitro settings and thus were missed in approaches based on in vitro-activated cells, validating our choice of in vivo models for expression profiling of myeloid cells.

Reactive oxygen species and 12/15-lipoxygenase contribute to the antiproliferative capacity of alternatively activated myeloid cells elicited during helminth infection.

Understanding the role of CD11b(+)GR-1(+) myeloid suppressor cells in the immune suppression and immunoregulation associated with a variety of diseases may provide therapeutic opportunities. In this article, we show, in a model of helminth infection, that CD11b(+)GR-1(+) myeloid suppressor cells but not CD11b(+)F4/80(high) mature macrophages expanded in the peritoneal cavity of BALB/c mice implanted with Taenia crassiceps. Peritoneal cell populations from early stage-infected animals impaired T cell proliferation by secreting NO. Yet, they lost their ability to secrete NO in the late stage of infection. Concomitantly, their capacity to exert arginase activity and to express mRNAs coding for FIZZ1 (found in inflammatory zone 1), Ym, and macrophage galactose-type C-type lectin increased. Furthermore, cells from early stage-infected mice triggered T cells to secrete IFN-gamma and IL-4, whereas in the late stage of infection, they only induced IL-4 production. These data suggest that CD11b(+)GR-1(+) myeloid suppressor cells displaying an alternative activation phenotype emerged gradually as T. crassiceps infection progressed. Corroborating the alternative activation status in the late stage of infection, the suppressive activity relied on arginase activity, which facilitated the production of reactive oxygen species including H(2)O(2) and superoxide. We also document that the suppressive activity of alternative myeloid suppressor cells depended on 12/15-lipoxygenase activation generating lipid mediators, which triggered peroxisome proliferator-activated receptor-gamma. IL-4 and IL-13 signaling contributed to the expansion of myeloid suppressor cells in the peritoneal cavity of T. crassiceps-infected animals and to their antiproliferative activity by allowing arginase and 12/15-lipoxygenase gene expression.

Macrophage galactose-type C-type lectins as novel markers for alternatively activated macrophages elicited by parasitic infections and allergic airway inflammation.

Molecular markers, especially surface markers associated with type II, cytokine-dependent, alternatively activated macrophages (aaMF), remain scarce. Besides the earlier documented markers, macrophage mannose receptor and arginase 1, we demonstrated recently that murine aaMF are characterized by increased expression of found in inflammatory zone 1 (FIZZ1) and the secretory lectin Ym. We now document that expression of the two members of the mouse macrophage galactose-type C-type lectin gene family (mMGL1 and mMGL2) is induced in diverse populations of aaMF, including peritoneal macrophages elicited during infection with the protozoan Trypanosoma brucei brucei or the Helminth Taenia crassiceps and alveolar macrophages elicited in a mouse model of allergic asthma. In addition, we demonstrate that in vitro, interleukin-4 (IL-4) and IL-13 up-regulate mMGL1 and mMGL2 expression and that in vivo, induction of mMGL1 and mMGL2 is dependent on IL-4 receptor signaling. Moreover, we show that expression of MGL on human monocytes is also up-regulated by IL-4. Hence, macrophage galactose-type C-type lectins represent novel surface markers for murine and human aaMF.

Secretory leukocyte protease inhibitor in cancer development.

Secretory leukocyte protease inhibitor (SLPI), an epithelial-specific serine protease inhibitor of the whey acidic protein (WAP) family, exerts broad tissue-protective functions: on the one hand, it counteracts inflammatory responses and invading pathogens, and on the other hand, it repairs the damaged tissue. Here, we report that subcutaneous in vivo passage of low-malignant 3LL-S cells increases the malignancy of these cancer cells. Applying the subtraction suppressive hybridization method to this cancer model, we identify SLPI as one of the genes whose level of expression directly correlates with the malignancy of the cancer cells. Using transfection experiments, we demonstrate that overexpression of mouse or human SLPI in 3LL-S cells is sufficient to enhance their malignant behavior, and that this activity of SLPI is related to its ability to inhibit serine proteases. Furthermore, using cDNA dot-blot hybridization, we show that in human gynecological cancer tissue SLPI expression is frequently increased as compared with normal tissue. This study underscores the promalignant role of SLPI in the development of cancer and its potential usefulness as a biomarker for gynecological cancer.