TLR agonists induce the differentiation of human bone marrow CD34+ progenitors into CD11c+ CD80/86+ DC capable of inducing a Th1-type response

We recently reported that human bone marrow hematopoietic CD34(+) progenitors express functional Toll-like receptors (TLR) and can differentiate into myeloid cells just by stimulation with resiquimod (R848), a specific agonist for TLR7/8. However, the mechanisms by which R848 induces cell differentiation, the effects of other TLR agonists and the functionality of the differentiated cells are not known. Comparable to R848, loxoribine (a TLR7 agonist) and Pam(3)CSK(4) (a TLR2 agonist) induced cytokine production and cell differentiation along the myeloid lineage. R848 and loxoribine were more effective than Pam(3)CSK(4) at inducing the lineage-negative (CD11c(+) CD14(-)) dendritic cells (DC), whereas Pam(3)CSK(4) was more effective at inducing CD11c(+) CD14(+) monocytes. Both cell subsets expressed CD80/CD86 and HLA-DR molecules; however, they showed differential expression of CD1a, CD1b, CD1c, CD11b, CD206 and CD207 markers when compared with each other. Cell differentiation into DC was significantly inhibited by an anti-TNF-alpha nonoclonal antibody. The CD11c(+) CD14(-) subset was isolated and shown to be more potent in stimulating an alloreaction than the CD11c(+) CD14(+) subset. Collectively, these data highlight the differential effects of TLR agonists on human bone marow CD34(+) progenitor cells and provide a new opportunity for generating functional DC that would be useful in cancer vaccination.     

Does our Current Understanding of Immune Tolerance,Autoimmunity, and Immunosuppressive Mechanisms Facilitate the Design of Efficient Cancer Vaccines?

The therapeutic use of the immune system to attack cancer cells has been a longstanding vision among tumour immunologists. However, most human tumours are poorly immunogenic and are able to invade the host immune system. Although these obstacles are clearly critical to cancer vaccine development, the induction of a strong anti-tumour immune response may rely on the activation of high affinity T cells through a molecular mimicry mechanism which involves cross-reactive recognition of foreign antigens mimicking the structure of tumour proteins. Taking into account the disparity in HLA molecules needed to present shared antigens; in late 1990s Stauss et al. described the possibility of generating allorestricted high affinity cytotoxic T cells against synthetic self-peptides bound to non-self-MHC molecules. In addition to the strategies indicated above, the inhibition of the immunosuppressive mechanisms associated with tumour invasion of the immune system using RNA interference also offers a new approach to vaccine design. This review highlights the problem of immune tolerance, the induction of autoreactive T cells, and describes strategies to enhance tumour immunity.     

Probing antinuclear antibody specificities by peptide phage display libraries

OBJECTIVE: To uncover the specificities of autoantibodies to nuclear proteins (ANA) in patients with juvenile rheumatoid arthritis (JRA). METHODS: Peptide ligands for ANA were selected by panning random peptide phage display libraries on antibodies binding to HEp-2 cells. Positive phage clones were identified by the immunoscreening technique. RESULTS: Groups of peptides were identified, some of which share the core motifs of KTTTnPY, RVADnL/I or RnNSPL. Perinuclear and nuclear staining of HEp-2 cells were obtained with patient serum antibodies binding to the phage displaying the core peptide motifs. In contrast, no significant reactivity was seen with the antibodies binding to the wild type phage. Antibodies to the phage displaying peptides containing some of the core motifs were detected more frequently in ANA-positive as compared to ANA-negative JRA patients. Homology search with the selected core motifs revealed a significant homology with a number of human nuclear proteins and proteins from potential infectious agents that could serve as trigger in the breakdown of tolerance. CONCLUSION: Panning of phage display libraries on antibodies reacting with cellular structures can lead to the identification of their specificities. Thus, the peptide epitopes reported here constitute additional information that may lead to the development of diagnostic tests and the identification of the parental antigens that initiated the B cell responses in patients with JRA.     

Identification of a novel centrosome/microtubule-associated coiled-coil protein involved in cell-cycle progression and spindle organization

Here we describe the identification of a novel vertebrate-specific centrosome/spindle pole-associated protein (CSPP) involved in cell-cycle regulation. The protein is predicted to have a tripartite domain structure, where the N- and C-terminal domains are linked through a coiled-coil mid-domain. Experimental analysis of the identified domains revealed that spindle association is dependent on the N-terminal and the coiled-coil mid domain. The expression of CSPP at the mRNA level was detected in all tested cell lines and in testis tissue. Ectopic expression of CSPP in HEK293T cells blocked cell-cycle progression in early G1 phase and in mitosis in a dose-dependent manner. Interestingly, mitosis-arrested cells contained aberrant spindles and showed impairment of chromosome congression. Inhibition of CSPP gene expression by small interfering RNAs induced cell-cycle arrest/delay in S phase. This phenotype was characterized by elevated levels of cyclin A, decreased levels of cyclin E and hyperphosphorylation of the S-phase checkpoint kinase Chk1. The activation of Chk1 may indicate a replication stress response due to an inappropriate G1/S-phase transition. Taken together, we demonstrate that CSPP is associated with centrosomes and microtubules and may play a role in the regulation of G(1)/S-phase progression and spindle assembly.     

Profiling the immune response in patients with breast cancer by phage-displayed cDNA libraries

Display on the surface of filamentous phages has been shown to be well suited for the enrichment of serum antibody-binding ligands. Here, we have taken the advantage of this technology to analyze the humoral immune response in patients with cancer. The cDNA repertoires from breast cancer cell lines T47D and MCF-7 were fused to the 3'-end of the filamentous phage M13 gene VI in all three reading frames. When the libraries were biopanned on rabbit polyclonal IgG against the human Bcl-x(L) protein, positive clones were selected, thus confirming the utility of the libraries. Using serum antibodies from patients with breast cancer, we specifically selected IgG-binding phage-encoded cDNA products. Sequence analysis of the selected clones identified important antigens including p53, centromere-F, int-2, pentraxin I, integrin beta5, cathepsin L2 and S3 ribosomal protein. The selected phage-displayed cDNA products were recognized by a significant number of breast cancer sera as compared to sera from normal individuals. Although the human pentraxin I mRNA was reported to be exclusively localized in the nervous system, we found it also expressed by breast cancer cell lines. Four out of 30 patients with breast cancer (13 %) showed reactivity with the recombinant pentraxin expressed in Escherichia coli, while no reactivity was found in normal sera. The obtained results demonstrate that phage display could be a valuable method for the identification of antigens recognized by the humoral immune system in patients with cancer.     

Macrocystic lymphatic lymphangioma (cystic lymphangioma) of the upper extremity: A case report

We report on a case of macrocystic lymphatic malformation of the forearm. A male infant, without any medical history, was followed up in our department since the age of 7 months because of a subcutaneous, soft, painless mass of the left forearm. Ultrasonography and the magnetic reasonance imaging (MRI) were evocative of a macrocystic lymphatic malformation. Five sessions of sclerotherapy led to the reduction of the size of the mass but another axillary tumor appeared afterwards. A surgical excision, unfortunately incomplete, was performed rapidly followed by a recurrence of the macrocystic lymphatic malformation. Macrocystic lymphatic malformations are localized in the neck in 75% and axilla in 20% of the cases. Involvement of the upper extremity and particularly the forearm is very rare. MRI is useful for the diagnosis and the definition of tumor limits. The treatment is usually challenging because of their location and rough delimitation.     

Colon cancer cell-derived tumor necrosis factor-alpha mediates the tumor growth-promoting response in macrophages by up-regulating the colony-stimulating factor-1 pathway

The interplay between malignant and stromal cells is essential in tumorigenesis. We have previously shown that colony-stimulating factor (CSF)-1, matrix metalloprotease (MMP)-2, and vascular endothelial growth factor (VEGF)-A production by stromal cells is enhanced by CSF-1-negative SW620 colon cancer cells. In the present study, the mechanisms by which colon cancer cells up-regulate host factors to promote tumorigenesis were investigated. Profiling of tumor cell cytokine expression in SW620 tumor xenografts in nude mice showed increased human tumor necrosis factor (TNF)-alpha mRNA expression with tumor growth. Incubation of macrophages with small interfering (si) RNAs directed against TNF-alpha or TNF-alpha-depleted SW620 cell conditioned medium versus SW620 cell conditioned medium failed to support mouse macrophage proliferation, migration, and expression of CSF-1, VEGF-A, and MMP-2 mRNAs. Consistent with these results, human TNF-alpha gene silencing decreased mouse macrophage TNF-alpha, CSF-1, MMP-2, and VEGF-A mRNA expression in macrophages cocultured with human cancer cells. In addition, inhibition of human TNF-alpha or mouse CSF-1 expression by siRNA reduced tumor growth in SW620 tumor xenografts in mice. These results suggest that colon cancer cell-derived TNF-alpha stimulates TNF-alpha and CSF-1 production by macrophages, and that CSF-1, in turn, induces macrophage VEGF-A and MMP-2 in an autocrine manner. Thus, interrupting tumor cell-macrophage communication by targeting TNF-alpha may provide an alternative therapeutic approach for the treatment of colon cancer.     

Genetic heterogeneity of megaloblastic anaemia type 1 in Tunisian patients

Megaloblastic anaemia 1 (MGA1) is a rare autosomal recessive condition characterized by selective intestinal vitamin B12 malabsorption and proteinuria. More than 200 MGA1 patients have been identified worldwide, but the disease is relatively prevalent in Finland, Norway and several Eastern Mediterranean regions. MGA1 is genetically heterogeneous and can be caused by mutations in either the cubilin (CUBN) or the amnionless (AMN) gene. In the present study we investigated the molecular defect underlying MGA1 in nine Tunisian patients belonging to six unrelated consanguineous families. Haplotype and linkage analyses, using microsatellite markers surrounding both CUBN and AMN genes, indicated that four out of the six families were likely to be linked to the CUBN gene. Patients from these families were screened for the Finnish, Mediterranean and Arabian mutations already published. None of the screened mutations could be detected in our population. One family showed a linkage to AMN gene. Direct screening of the AMN gene allowed the identification of the c.208-2A>G mutation, previously described in a Jewish Israeli patient of Tunisian origin and in Turkish patients. This suggests that the c.208-2A>G mutation may derive from a single Mediterranean founder ancestor. For the last family, haplotype analysis excluded both CUBN and AMN genes, suggesting the existence of a third locus that may cause MGA1.     

Antitumoral Potency by Immunomodulation of Chloroform Extract from Leaves of Nitraria retusa,Tunisian Medicinal Plant,via its Major Compounds β-sitosterol and Palmitic Acid in BALB/c Mice Bearing Induced Tumor

This study evaluated the antitumoral effect of Chloroform extract from Nitraria retusa leaves, via its major compounds β-sitosterols and palmitic acid. BALB/c mice were subcutaneously inoculated with B16-F10 cells, then treated intra-peritoneally after 7 days with the chloroform extract for 21 days. They were then euthanized, and the tumors were weighed. Lung parenchyma was analyzed. Lymphocyte and macrophages proliferation, cytotoxic T lymphocyte (CTL) activities were evaluated using the MTT assay. Macrophage phagocytosis was evaluated by measuring the lysosomal activity and nitric oxide production. Antioxidant activity was studied by cellular antioxidant activity on macrophage and splenocytes and by lipid peroxidation inhibitory activity in liver cells, kidney, and serum.

β-sitosterols and palmitic acid, major compounds of chloroform extract, impeded remarkably the expansion of the transplantable tumor, protected the lung parenchyma, and increased splenocytes proliferation and both CTL activities in tumor-bearing mice. β-sitosterols and palmitic acid were also seen to have enhanced lysosomal activity of host macrophages and antioxidant cellular activity. Also, they showed an inhibitory effect of lipid peroxidation.

Our results suggest that antitumoral effect of β-sitosterols and palmitic acid from chloroform extract is related with its immunomodulatory activity, and opens the way for a nutrition application and coprocessing phytotherapy against cancer.