Guillermo A. Blanco

Hyaluronan oligosaccharides sensitize lymphoma resistant cell lines to vincristine by modulating P-glycoprotein activity and PI3K/Akt pathway

Multidrug resistance (MDR) is one of the main reasons for failure of cancer therapy. It may be mediated by overexpression of ATP‐dependent efflux pumps or by alterations in survival or apoptotic pathways. Fragments generated by enzymatic degradation of hyaluronan (oHA) were able to modulate growth and cell survival and sensitize MDR breast cancer cells to cytotoxic drugs. In this work the relationship between oHA and MDR in lymphoid malignancies was analyzed using murine lymphoma cell lines resistant to doxorubicin (LBR‐D160) or vincristine (LBR‐V160) and a sensitive line (LBR‐). After oHA treatment, higher apoptosis levels were observed in the resistant cell lines than in the sensitive one. Besides, oHA sensitized LBR‐D160 and LBR‐V160 to vincristine showing increased apoptosis induction when used in combination with vincristine. Native hyaluronan failed to increase apoptosis levels. As different survival factors could be modulated by hyaluronan, we investigated the PI3K/Akt pathway through PIP3 production and phosphorylated Akt (p‐Akt) and survivin expression was also evaluated. Our results showed that oHA decreased p‐Akt in the 3 cell lines while anti‐CD44 treatment abolished this effect. Besides, survivin was downregulated only in LBR‐V160 by oHA. When Pgp function was evaluated, we observed that oHA were able to inhibit Pgp efflux in murine and human resistant cell lines in a CD44‐dependent way. In summary, we report for the first time that oHA per se modulate MDR in lymphoma cells by decreasing p‐Akt as well as Pgp activity, thus suggesting that oHA could be useful in combination with classical chemotherapy in MDR hematological malignancies.

Interaction of CD44 with Different Forms of Hyaluronic Acid. Its Role in Adhesion and Migration of Tumor Cells

Interaction between hyaluronic acid (HA) and CD44 has been considered a key event in tumor invasion and metastasis. HA is a linear, high molecular weight glycosaminoglycan in its native state, but fragmented low molecular forms are found at sites of neoplastic or inflammatory infiltrates. Both high and low molecular weights HA are involved in diverse biological functions. In this study, we used two clonal variants of a T cell murine lymphoma designated LBLa and LBLc. These cell lines were found to differ in their in vivo and in vitro growth rates. LBLa grew faster and exhibited an enhanced invasive capacity as compared to LBLc. In contrast, cell lines did not differ in the expression of surface markers (CD8, CD24, CD25, CD44, and CD18), or in their capacity to bind HA. However, LBLa cells exhibited higher capacity to migrate to low molecular weight HA than did LBLc. Migration was mediated by CD44 since it was abrogated by anti-CD44 monoclonal antibody as well as by hyaluronidase. We suggest that interaction between CD44 and low molecular weight HA may trigger migration mechanisms in LBLa cells, thus contributing to enhanced invasive cell capacity.

LPS-induced stimulation of phagocytosis in the sipunculan worm Themiste petricola: possible involvement of human CD14, CD11B and CD11C cross-reactive molecules.

Coelomocytes of Themiste petricola, a marine invertebrate of the phylum Sipuncula, were exposed in vitro to bacterial lipopolysaccharides (LPS), and the phagocytic activity against heat-killed yeast (Saccharomices cerevisiae) was evaluated using a flow cytometric assay. An increase of phagocytic activity was observed following pre-incubation of coelomocytes over 20 h with either 5 micrograms/mL LPS or 1.5 micrograms/mL phorbol 12-myristate 13-acetate (PMA). The phagocytic enhancement induced by LPS was blocked by co-incubation with polymixin B, a ligand for the lipid A region of LPS. In a 72 h stimulation assay, LPS was also found to enhance phagocytosis. The enhancement was significantly higher when coelomocytes were incubated with LPS plus coelomic plasma. Using mAbs directed against human CD14 and components of the human LFA-1 complex, we identified coelomocyte surface antigens cross-reactive with CD14, CD11b and CD11c. The expression of CD11b and CD11c antigens was augmented by LPS treatment of coelomocytes. By double fluorescence assays, using mAb Leu-M3 and fluorescein labeled yeast, phagocytic coelomocytes were found to be mainly anti-CD14 positive. No cross-reactions were detected with mAbs against CD11a and CD18. Enzymatic treatment of coelomocytes with phosphatidyl inositol phospholipase C (PI-PLC) reduced the expression of the CD14-like antigen. The presence, in sipunculan coelomocytes, of antigens cross-reactive with CD14, the alpha chain of CR3 and of p150,95 raises the possibility that molecules related, although not necessary homologous, to the mammalian counterparts may have a role in the defense systems of these animals.

Trichinella infection in wild boars (Sus scrofa) from a protected area of Argentina and its relationship with the presence of humans

In Argentina, Trichinella infection has been documented in humans and animals of several provinces since 1930. This zoonotic parasite infection has been recently detected in humans and pigs of a region historically considered as Trichinella-free, suggesting the spread of these pathogens. The aim of the present work was to investigate the presence of Trichinella infection in wild boars (Sus scrofa) and in the human population living in a protected area. Trichinella infection has been investigated by serology (in humans and wild boars) and by artificial digestion of wild boar muscles. The isolated Trichinella larvae have been identified at the species level by multiplex PCR. A geographical information system has been used to collect environmental data. The results showed the circulation of Trichinella spiralis in wild boars with a low parasite burden, and suggest the influence of human behavior on the transmission. The transplacental passage of parasite is postulated. It follows that the declaration of region as Trichinella-free should be carefully established by means of extensive monitoring programs, not only in humans and domestic animals but also in wildlife.

Immune systems, geographic information systems (GIS), environment and health impacts.

Exposure to dioxins, polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) has been related to alterations in cellular and humoral immune responses in both adaptive and innate immune systems of most animal species. These compounds share a common signaling mechanism to exert their effects on cells of the immune system, which includes the aryl-hydrocarbon receptor (AhR) and the AhR nuclear translocator (ARNT). Recently, the interference of AhR–ARNT with the nuclear factor (NF)-κB signaling pathway has been proposed as a critical event in the adverse effects on the immune system. Studies on the effects of these AhR–ARNT-related toxicants on the immune system of higher and lower phylum animals and knowledge of intracellular mechanisms of toxicity may contribute to development of biomarkers of ecotoxicant exposure and effects. Biomarkers of this kind allow sampling over extended geographic areas, in several sentinel species, including wildlife animals, and facilitate the building of risk models and risk maps of environmentally induced diseases. On the basis of location, biomarker sampled data obtained through evaluation of ecotoxicant exposure and effects on the immune system in sentinel species can be further integrated and analyzed together with other sources of environmental geographic information, or human population health data, by means of geographic information systems (GIS). The spatial analysis capability of GIS can help to evaluate the complex relationships of overlaid information and to identify areas with high risk indices or “hot spots.” This integrative approach can be useful in studies contributing to support environmental and health-related policies and regulations.

Hydrogen Peroxide Induces Apoptotic-Like Cell Death in Coelomocytes of Themiste petricola (Sipuncula)

Apoptosis is an active form of cell death that plays a critical role in physiological and pathological conditions of multicellular organisms. These conditions include development, organogenesis, and elimination of infected, mutated, or damaged cells. Sipunculan cells may respond to changes in environmental exposure to oxidative stress by induction of apoptotic cell death. In coelomocytes of the sipunculan worm Themiste petricola, we evaluated morphological and biochemical changes that were induced by hydrogen peroxide (H2O2) and that could be compatible with an apoptotic-like phenotype. At an exposure of 100 mM H2O2, coelomocytes exhibited several morphological hallmarks of apoptosis such as chromatin condensation, nuclear segmentation, cell volume decrease, membrane blebbing, and formation of apoptotic bodies. Biochemical evidences of apoptotic-like cell death included exposure of phosphatidylserine (PS) in the outer leaflet of the plasma membrane and oligonucleosomal DNA fragmentation. In addition, exposure of coelomocytes to H2O2 induced a rapid massive loss of mitochondrial membrane potential and of the acidic pH of lysosomes. Overall, our results showed that, in sipunculan coelomocytes, H2O2 can induce changes compatible with an apoptotic-like phenotype. The finding of an oxidative-stress-induced apoptotic-like phenotype in a sipunculan worm may indicate that this kind of cell death process participates in regulation of cell number during physiological and pathological situations, including immune responses.

Hyaluronan induces migration of multidrug-resistant lymphoma cell lines in vitro through Tiam1 activation by a PI3K-dependent mechanism

Hyaluronan (HA) modulates multidrug resistance (MDR) as well as cell migration. Tiam1 is involved in cytoskeleton reorganization during tumor invasion. In this report we show the relationship among HA, Tiam1, migration and MDR in murine lymphoma cell lines. We observed that MDR cells presented higher migratory capacity towards HA in vitro as well as higher constitutive active Tiam1 expression than the sensitive cell line. Besides, HA treatment induced migration towards HA of MDR cell lines through Tiam1 activation by a PI3K-dependent mechanism, showing that disruption of HA signaling would be useful in treatment of MDR hematological malignancies.

Coelomocyte locomotion in the sipunculan Themiste petricola induced by exogenous and endogenous chemoattractants: role of a CD44-like antigen–HA interaction

Cell migration is a key event in the invertebrate immuno-defense system. Microbial products like lipopolysacharide (LPS) and formyl-methyl-leucyl-phenylalanine (fMLP) promote cell recruitment to sites of infection. In mammals, complement activation by factors such as zymosan induces C5a production, which influences leukocyte migration. The endogenous factor hyaluronic acid (HA), an extracellular matrix component, also promotes cell migration through its receptor CD44. We evaluated whether coelomocytes from the sipunculan worm T. petricola migrated towards LPS, fMLP, or zymosan treated plasma (ZTP) and if HA was involved in coelomocyte migration and adhesion. We also evaluated if antibodies specific for mouse HA receptor CD44 inhibited any of the effects induced by HA. Using microchemotaxis chambers we found that coelomocytes migrated towards exogenously and endogenously derived chemoattractants. We also observed that HA was a potent chemotactic signal and that coelomocytes adhered strongly to plates coated with LMW-HA but not with HMW-HA. In addition we found that these HA mediated effects were blocked by the monoclonal antibody IM7 directed to mouse CD44, suggesting that a CD44-like cross-reactive antigen might play a role in HA mediated coelomocyte locomotion.

Sublethal infection with Salmonella Enteritidis by the natural route induces intestinal and joint inflammation in mice

Reactive arthritis (ReA) is a sterile inflammation triggered by a distal mucosal infection, which suggests a contribution from bacterial products. Investigation on the pathogenesis of ReA is difficult because of the limited studies that can be performed in humans; therefore the availability of animal models is crucial. We hereby describe a murine model for studying the early stages of Salmonella-induced ReA. BALB/c mice infected by the natural route with a sublethal dose of S. Enteritidis showed long lasting gut inflammation, synovitis in the knee joint and a significant increase of CD4+ lymphocytes in the draining popliteal lymph nodes. S. Enteritidis infection induced histological changes in intact knees and exacerbated inflammation in previously damaged joints. Experiments performed with S. Enteritidis DeltainvG mutant suggest that the proinflammatory signalling mediated by Salmonella TTSS-1 in the gut is required for the induction of joint sequelae. Since this model is highly reproducible and easy to perform, it provides great potential for investigating both host and bacterial contributions to the early stages of ReA.

Caffeic acid phenylethyl ester and MG132, two novel nonconventional chemotherapeutic agents, induce apoptosis of human leukemic cells by disrupting mitochondrial function

The ability to modulate balance between cell survival and death is recognized for its great therapeutic potential. Therefore, research continues to focus on elucidation of cell machinery and signaling pathways that control cell proliferation and apoptosis. Conventional chemotherapeutic agents often have a cytostatic effect over tumor cells. New natural or synthetic chemotherapeutic agents have a wider spectrum of interesting antitumor activities that merit in-depth studies. In the present work, we aimed at characterizing the molecular mechanism leading to induction of cell death upon treatment of the lymphoblastoid cell line PL104 with caffeic acid phenylethyl ester (CAPE), MG132 and two conventional chemotherapeutic agents, doxorubicine (DOX) and vincristine (VCR). Our results showed several apoptotic hallmarks such as phosphatidylserine (PS) exposure on the outer leaflet of the cell membrane, nuclear fragmentation, and increase sub-G1 DNA content after all treatments. In addition, all four drugs downregulated survivin expression. CAPE and both chemotherapeutic agents reduced Bcl-2, while only CAPE and MG132 significantly increased Bax level. CAPE and VCR treatment induced the collapse of mitochondrial membrane potential (∆ψm). All compounds induced cytochrome c release from mitochondrial compartment to cytosol. However, only MG132 caused the translocation of Smac/DIABLO. Except for VCR treatment, all other drugs increased reactive oxygen species (ROS) production level. All treatments induced activation of caspases 3/7, but only CAPE and MG132 led to the activation of caspase 9. In conclusion, our results indicate that CAPE and MG132 treatment of PL104 cells induced apoptosis through the mitochondrial intrinsic pathway, whereas the apoptotic mechanism induced by DOX and VCR may proceed through the extrinsic pathway.