The effects of some Curdlan derivatives on Dectin-1 expression and cytokine production in human peripheral blood mononuclear cells.

The cells of immune system such as monocytes and macrophages are in first line defence against dangerous signals. In the present paper the recognition of Dectin 1 receptors and the modulation of Interleukin-10 (IL-10) and Tumor Necrosis Factor-alpha (TNF-alpha) cytokine production by Curdlan and Curdlan derivatives in peripheral blood mononuclear cells (PBMCs) were studied. The effect of Curdlan or Curdlan derivatives on the expression of Dectin 1 receptors in PBMCs was revealed by flow-cytometry and the levels of IL-10 and TNFalpha were measured by ELISA kit in supernatants of PBMCs cultured in presence or absence of Curdlan, Curdlan derivatives and LPS. Our results suggested that Curdlan and Curdlan derivatives were able to increase the expression of Dectin-1 receptors on monocyte cells. The combined treatment of Curdlan/Curdlan derivatives and Pam3Cys produced an increase of CD14+ cells possessing Dectin-1 receptors. We demonstrated that Curdlan (at 20 microg unique dose) up-regulated TNF-alpha production and down-regulated IL-10 production in PBMCs. Conversely, Palm CM/SP-Curdlan (20 microg unique dose) was able to down-regulate TNF-alpha production and to up-regulate IL-10 production in PBMCs. For instance, Palm CM/SP-Curdlan determined a 5 times decrease of TNF-alpha production than Curdlan. Regarding the effect of Palm CM/SP-Curdlan on IL-10 production in PBMCs, we noticed that the level of IL-10 was about 4 times greater than Curdlan activity. We observed that a combined treatment of Curdlan/Curdlan derivatives and LPS induced about 5 times decrease in TNF-alpha production in PBMCs. IL-10 production induced by Palm CM/SP-Curdlan and LPS was about 6 times greater than the combined effect of Curdlan and LPS. The treatment of PBMCs with SP-Curdlan alone affected neither TNF-alpha production nor IL-10 production. Our results are in accordance with other studies demonstrating that Dectin-1 and TLR2/TLR6 signaling combine to enhance the responses triggered by each receptor and the signaling pathway induced by Dectin-1 could mediate the production of pro-inflammatory cytokines.

Curdlan derivatives able to enhance cytostatic drugs activity on tumor cells.

The chemotherapy success to kill cancer cells depends on its ability to stop cell division. The faster the cells are dividing, the more likely it is that chemotherapy will kill the cells, causing the tumor to shrink. Taking into account the severe side effects of chemotherapy, drugs producers also focus on natural products obtained either from medicinal plants, or from microorganisms. The complex polysaccharides named beta-glucans are active compounds with immune activity. beta-glucan polymers belong to a class of drugs with effects on the immune system, such as: anti-tumoral, anti-infectious, protection against fungi, bacteria and viruses infections. The correct selection of beta-glucans is essential to identify compounds with favorable clinical effects. The aim of this study was to investigate the capacity of six Curdlan (beta-glucan) derivatives to up-regulate the Doxorubicin, Actinomycin D and Cyclophophamide cytostatic drug activity on tumor cells (murine B16 melanoma and human HEp-2 laryngeal carcinoma cell lines). Our results demonstrated that Palm SP derivative, as well as SP and Palm CM/SP derivatives were able to potentiate Doxorubicin action or Actinomycin D effect on B16 tumor cells. SP derivative significantly enhanced cytostatic activity of Cyclophosphamide on B16 cells. All the investigated Curdlan derivatives (SP, Palm CM/SP, CM/SP, Palm CM, Palm SP and CM) were able to inhibit HEp-2 tumor cell growth, by up-regulating Doxorubicin and Actinomycin D cytostatic activity.

The modulation of reactive oxygen species production from human polymorphonuclear cells by curdlan derivatives as dectin-1 agonists/antagonists.

Reactive oxygen species (ROS) are well known to be cytotoxic and have been implicated in the etiology of a wide array of human diseases including diabetes, neurodegenerative diseases, cancer and also influence central cellular processes such as proliferation, apoptosis, senescence etc. If in these pathological or degenerative conditions characterized by free radicals excess, reactive species are not eliminated, they can maintain destructive processes, already initiated at different cellular levels. Understanding the role of ROS as key mediators in signaling cascades may provide various opportunities for pharmacological intervention. Toll-like receptors and C-type lectin receptor class V--Dectin-1, as members of Pattern Recognition Receptors play an essential role in innate immune response against bacteria and fungi respectively, contributing to pathogens recognition, phagocytosis, ROS production and induction of pro-inflammatory cytokines secretion. Using a high performance chemiluminometric method, we studied the action of six Curdlan derivatives on the ROS production and release by activated human polymorphonuclear cells (PMNs) isolated from the peripheral blood of healthy donors. Our results demonstrated that Curdlan derivatives containing sulfopropyl groups did not activate human PMNs to release ROS. These compounds blocked Dectin-1 and were able to inhibit co-operation between Dectin-1 and TLR-2. Curdlan derivatives containing palmithoyl, carboxi-methyl and sulfopropyl groups increased ROS release by human PMNs activated at TLR-2 level. Taking into account the fact that Dectin-1 can actively collaborate with TLR-2 to modulate the subsequent adaptive immune response, we can presume that Curdlan derivatives containing sulfopropyl group or palmithoyl/carboxi-methyl/sulfopropyl groups, as possible Dectin-1 antagonists/agonists, could influence TLR-2 signaling.

Recognition and modulation of Dectin-1 and TLR-2 receptors by curdlan derivatives and purified natural extracts.

Toll-like receptors (TLRs) and Dectin-1, as members of Pattern Recognition Receptors play an essential role in innate immune response against bacteria and fungi respectively, contributing to pathogens recognition, phagocytosis, etc. Dectin-1 and TLR-2/TLR-6 can interact for intracellular signal transduction. Dectin-1 is expressed at low levels on macrophages and at high levels on dendritic cells. Dectin-1 and TLRs are synergistic in mediating cytokines production, such as IL-12 and tumor necrosis factor alpha (TNF alpha). In the present paper we studied the expression of Dectin-1 (beta-Glucan Receptor C-type lectin receptor class V) and TLR-2 on human normal monocytes cells and also the role of different Curdlan derivatives and highly purified natural extracts, especially their capacity to recognize these receptors and their Dectin-1 agonist/antagonist properties. Our results demonstrated that Curdlan derivatives containing sulfopropyl or palmythoil/carboximethyl/sulfopropyl groups and natural extracts could be potent immunomodulators with many potential applications (possible antagonists of Dectin-1, blockers of Dectin-1 cooperation with TLR-2).

BM2L is a spontaneous leukemogenic variant of a non-leukemogenic v-myb-transformed myeloid cell line.

Leukemogenesis is a complex process involving an accumulation of genetic lesions affecting both growth and differentiation in cells of the hematopoietic lineage. Our laboratory has established a non-producer v-myb-transformed cell line (BM2/C3A) which, when injected into the chicken embryo, does not produce leukemia. Recently, a spontaneous variant of this cell line, called BM2L, was obtained from in vivo experiments. BM2L produces an acute monoblastic leukemia when injected into the chicken embryo. BM2L cells do not differentiate in vivo or in vitro, but continue to proliferate under conditions in culture that allow for the differentiation of BM2/C3A cells into macrophages. In addition, BM2L cells have reduced requirements for exogenous growth factors. BM2L cells contain the v-myb allele and express v-Myb protein, but leukemogenicity does not involve point mutations in v-myb. The BM2 model, consisting of two non-producer cell lines differing in vivo in their leukemogenicity, provides a novel system for identifying genes that play a role in the induction or suppression of leukemogenesis.

Monoclonal antibodies recognizing normal and retrovirus-transformed chicken hematopoietic cells.

The avian hematopoietic system has long been an invaluable model to study the mechanisms of cell growth and differentiation. We have developed six MAbs against either chicken embryonic hematopoietic precursor cells or retrovirus-transformed cells. MAbs Mo1, Mo2, and Mo3 recognized transformation-associated markers expressed in AMV-transformed nonproducer cell line-BM2. Not only were these markers expressed 7 to 10 folds higher on BM2 than on normal monocytic cells, but their expression was drastically reduced when BM2 cells were induced to differentiate into macrophages by PMA. The control of marker expression is associated with v-myb-transforming cascade, since another monocytic lineage-specific oncogene, v-myc, did not enhance the expression of these markers. MAb Em1 detected a marker that is normally present in 20% of the cells from the 30/50% interface of a discontinuous percoll gradient of normal 4-day-embryo yolk sac. Its expression is also found in AEV-transformed cells and MSB1 cells. The epitope for Em1 was exposed after neuraminidase treatment on erythroleukemia cell line 6C2, which suggested that sialylation and/or glycosylation is pivotal in regulating the expression of specific markers in differentiation pathways during embryogenesis and tumorigenesis. MAb Em2 recognized proliferating hematopoietic cells after the fourth day of embryogenesis. MAb Em3, on the other hand, is presumed to be specific for an oncofetal antigen expressed in various transformed cells but only in 10% of the cells from 30/50% interface of a discontinuous percoll gradient of normal 4-day-embryo yolk sac. These MAbs will be useful for dissecting the expression of differentiation markers within normal versus abnormal differentiation pathways in molecular terms.

EGF-R as a hemopoietic growth factor receptor: the c-erbB product is present in chicken erythrocytic progenitors and controls their self-renewal.

c-erbB, encoding the EGF receptor (EGF-R), was originally identified as the cellular homolog of a chicken leukemia oncogene. In humans, EGF-R is distributed widely except in hemopoietic tissues, and its amplification is associated with epidermal and glial malignancies. Here we show that c-erbB is present in normal chicken erythrocytic progenitors and transmits the mitogenic signal induced by TGF alpha. Cells that contain high affinity EGF-R are at approximately the BFU-E stage, and their long-term renewal can be induced by TGF alpha. Upon addition of insulin and erythropoietin, they can be induced to terminally differentiate into red cells. We previously demonstrated that v-erbA blocks differentiation of chicken erythrocytic progenitors but does not abrogate their growth factor dependence for proliferation. These data indicate that proliferation and differentiation are not necessarily coupled in these cells. They also demonstrate a direct role of c-erbB in the control of self-renewal of normal chicken erythrocytic progenitors and could account for the predominant leukemogenic potential of the chicken erbB gene.

Expression of the v-erbA product, an altered nuclear hormone receptor, is sufficient to transform erythrocytic cells in vitro.

We investigated the effect of the v-erbA oncogene product, an altered thyroid hormone receptor, in chicken erythrocyte progenitor cells. Bone marrow cells were infected with a retrovirus vector (XJ12) carrying the v-erbA gene in association with the neoR gene. XJ12-infected erythrocyte progenitor cells gave rise to G418-resistant clones. Some were composed of blast cells identified as transformed CFU-Es blocked in their differentiation. These cells could be grown in culture for at least 25 generations and required anemic chicken serum as a source of erythropoietic growth factors. XJ12 can infect erythrocyte progenitor cells in vivo but is not sufficient to induce erythroleukemia. These data suggest that the activation of a nuclear hormone receptor might represent one step toward the development of neoplasms.

Avian erythroblastosis virus transforms a novel mast cell-basophil precursor target in the Japanese quail.

Hematopoietic cells of the Japanese quail were transformed by avian erythroblastosis virus in vivo and in vitro. In both circumstances, the infected hematopoietic tissues exhibited a dual oncogenic response of erythroid and mast cell-basophil elements. The erythroid transformants escaped the avian erythroblastosis virus block in differentiation and progressed to hemoglobinization. Resulting basophilic cells were morphologically, biochemically, and ultrastructurally identical to mast cell-basophils observed in other species. None of the virally transformed cells actively produced reverse transcriptase activity. Nonproducer cell lines synthesized viral RNA and both v-erbA and v-erbB proteins. These results indicate that the Japanese quail has a viral target cell different from that of the chicken. The implications of a single bipotential transformation target yielding both erythroid and mast cell-basophil colonies is discussed.

Molecular characterization of three erbB transducing viruses generated during avian leukosis virus-induced erythroleukemia: extensive internal deletion near the kinase domain activates the fibrosarcoma- and hemangioma-inducing potentials of erbB.

Three new erbB transducing viruses generated during avian leukosis virus-induced erythroblastosis have been cloned and sequenced, and their transforming abilities have been analyzed. Provirus 9134 E1 expresses an amino-terminally truncated erbB product that is analogous to the proviral insertionally activated c-erbB gag-erbB fusion product. This virus efficiently induces erythroblastosis, but does not transform fibroblasts in vitro or induce sarcomas in vivo. In contrast, virus 9134 S3 expresses an erbB product identical to the erbB product of 9134 E1, with the exception of a large internal deletion located between the kinase domain and the putative autophosphorylation site, P1. Interestingly, this virus is no longer capable of inducing erythroblastosis, but can induce both fibrosarcomas and hemangiomas in vivo. Provirus 9134 F3 has sustained an approximately 23-amino-acid carboxy-terminal truncation and is capable of inducing both erythroblastosis and sarcomagenesis. This virus expresses an erbB product with the shortest carboxy-terminal truncation sufficient to reveal the sarcomagenic potential of this protein. The distinct transforming properties of these viruses indicate that different structural domains of the erbB product confer distinct disease specificities.

Transformation-defective mutant of avian myeloblastosis virus that is temperature sensitive for production of transforming protein p45v-myb.

We have characterized a mutant of avian myeloblastosis virus (strain GA907/7) that shows a reduced capacity to transform myelomonocytic cells at the nonpermissive temperature. Myeloblasts transformed by this mutant suffer a substantial decrease in the amount of the transforming protein p45v-myb when shifted from the permissive to the nonpermissive temperature. We presume that the 5- to 10-fold decrease in the amount of p45v-myb causes the loss of the transformed phenotype. The decrease is due to a reduction in the level of v-myb mRNA. Mutant GA907/7 thus provides genetic evidence that p45v-myb is the transforming protein of avian myeloblastosis virus and apparently represents an unusual defect in the production or stability of mRNA.

Embryonic erythroid cells transformed by avian erythroblastosis virus may proliferate and differentiate.

Embryonic chick cells from the primitive streak stage to later stages of the developing embryo were infected with avian erythroblastosis virus (AEV). The data indicate that the greatest number of target cells for AEV was observed in the 12-somite blastoderm and gradually decreased in hemopoietic tissues with the development of the embryo. The target cell for AEV is not in the BFU-E compartment, as it is in the adult bone marrow, but is probably recruited within the CFU-M compartment which precedes the BFU-E compartment. Our studies also show that a significant number of transformed colonies derived from embryonic hemopoietic tissues undergo hemoglobinization in contrast with what is observed in transformed colonies of bone marrow. A complete characterization of the embryonic and adult hemoglobin is at present under study.

Characterization of the hemopoietic target cells for the avian leukemia virus E26.

The dual leukemogenic response, involving both the erythroid and myeloid hemopoietic systems in chickens infected with E26 virus, has previously been described (C. Moscovici, J. Samarut, L. Gazzolo, and M. G. Moscovici, 1981. Virology 113, 765-768; K. Radke, H. Beug, S. Kornfeld, and T. Graf, 1982. Cell 31, 643-653). Similarly, the in vitro response of the two lineages resulted in the concomitant transformation and proliferation of erythroblast and myeloblast leukemic cells. The present study, using embryonic tissues at very early stages of development, was valuable in implying that E26 target cells are recruited among uncommitted erythroid-myeloid stem cells as well as myeloid- or erythroid-committed progenitor cells. Therefore, E26 may be the first avian retrovirus capable of interacting with uncommitted hemopoietic precursor cells.

The product of the retroviral transforming gene v-myb is a truncated version of the protein encoded by the cellular oncogene c-myb.

Avian myeloblastosis virus (AMV) is an oncogenic retrovirus that rapidly causes myeloblastic leukemia in chickens and transforms myeloid cells in culture. AMV carries an oncogene, v-myb, that is derived from a cellular gene, c-myb, found in the genomes of vertebrate species. We constructed a plasmid vector that allows expression of a portion of the coding region for v-myb in a procaryotic host. We then used the myb-encoded protein produced in bacteria to immunize rabbits. The antisera obtained permitted identification of the proteins encoded by both v-myb and chicken c-myb. The molecular weights of the products of v-myb and c-myb (45,000 and 75,000 respectively) indicate that the v-myb protein is an appreciably truncated version of the c-myb protein.

Isolation and characterization of a temperature-sensitive mutant of avian myeloblastosis virus.

A temperature-sensitive (ts) mutant, GA 907/7, was isolated after mutagen treatment of avian myeloblastosis virus. When bone marrow cells or secondary yolk sac macrophages were infected with GA 907/7, the expression of transformation was greatly reduced at 41 degrees C. The results of temperature-shift experiments suggest that in GA 907/7 the putative v-myb gene product is functional only at 35.5 degrees C. Moreover, when ts-induced transformed cells were shifted to 41 degrees C, a partial morphological conversion to macrophage-like cells was obtained, while the majority of the cells underwent senescence and lysis. No leukemia was obtained when GA 907/7 was injected in 1-day-old chickens. Finally, a continuous cell line releasing genetically stable mutant virus was obtained after transformation of secondary yolk sac cells.

Transforming ability of avian defective leukemia viruses in early embryogenesis.

The response to infection of chicken hemopoietic cells derived from the early stages of embryogenesis by avian myeloblastosis virus (AMV) and avian erythroblastosis virus (AEV) was investigated. It was found that erythroid progenitor cells were present in the blastoderm at a higher frequency than that of myeloid progenitor cells. These results correlate with the observation that target cells for AEV were found to be more numerous than those for AMV. Therefore, blastoderm cells are of potential value in understanding the mechanisms of oncogenesis at the level of the target cells.

The genome and the intracellular RNAs of avian myeloblastosis virus.

Avian myeloblastosis virus (AMV) is an acute leukemia virus which causes a myeloblastic leukemia in birds and transforms myeloid hematopoietic cells in vitro. We have analyzed RNA from AMV virions and from AMV-transformed producer and nonproducer cells by gel electrophoresis followed by transfer to chemically activated paper and hybridization to several complementary DNA (cDNA) probes. Using a cDNA probe specific for AMV, we identified two RNA species of 7.2 and 2.3 kb, which were present in all AMV-transformed cells and in all AMV virion preparations examined. The 7.2 kb species, which is presumably the genome of AMV, appears to contain the entire retroviral gag gene and at least part of the pol gene, but lacks much (or all) of the env gene. Thus AMV differs from other acute leukemia viruses described to date, since the latter have genomes of 5.5 to 5.6 kb, have only part of the gag gene and lack pol sequences. The smaller RNA does not contain gag-, pol- or env-specific nucleotide sequences but does carry nucleotide sequences from both the 5' and 3' termini of the genome, suggesting that it may be a subgenomic mRNA. Both the 7.2 and 2.3 kb species were associated with the 70S RNA complex in virions. These results suggest that AMV, unlike other acute leukemia viruses, does not express its transforming gene via a gag-related "fusion" protein but rather as a (so far unidentified) protein translated from a subgenomic mRNA.