Gabriel A. Rabinovich

Galectin 3 Induces a Distinctive Pattern of Cytokine and Chemokine Production in Rheumatoid Synovial Fibroblasts via Selective Signaling Pathways

OBJECTIVE High expression of galectin 3 at sites of joint destruction in rheumatoid arthritis (RA) suggests that galectin 3 plays a role in RA pathogenesis. Previous studies have demonstrated the effects of galectins on immune cells, such as lymphocytes and macrophages. This study was undertaken to investigate the hypothesis that galectin 3 induces proinflammatory effects in RA by modulating the pattern of cytokine and chemokine production in synovial fibroblasts. METHODS Matched samples of RA synovial and skin fibroblasts were pretreated with galectin 3 or tumor necrosis factor alpha (TNFalpha), and the levels of a panel of cytokines, chemokines, and matrix metalloproteinases (MMPs) were determined using enzyme-linked immunosorbent assays and multiplex assays. Specific inhibitors were used to dissect signaling pathways, which were confirmed by Western blotting and NF-kappaB activation assay. RESULTS Galectin 3 induced secretion of interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor, CXCL8, and MMP-3 in both synovial and skin fibroblasts. By contrast, galectin 3-induced secretion of TNFalpha, CCL2, CCL3, and CCL5 was significantly greater in synovial fibroblasts than in skin fibroblasts. TNFalpha blockade ruled out autocrine TNFalpha-stimulated induction of chemokines. The MAPKs p38, JNK, and ERK were necessary for IL-6 production, but phosphatidylinositol 3-kinase (PI 3-kinase) was required for selective CCL5 induction. NF-kappaB activation was required for production of both IL-6 and CCL5. CONCLUSION Our findings indicate that galectin 3 promotes proinflammatory cytokine secretion by tissue fibroblasts. However, galectin 3 induces the production of mononuclear cell-recruiting chemokines uniquely from synovial fibroblasts, but not matched skin fibroblasts, via a PI 3-kinase signaling pathway. These data provide further evidence of the role of synovial fibroblasts in regulating the pattern and persistence of the inflammatory infiltrate in RA and suggest a new and important functional consequence of the observed high expression of galectin 3 in the rheumatoid synovium.

Characterization and functionality of proliferative human Sertoli cells.

It has long been thought that mammalian Sertoli cells are terminally differentiated and nondividing postpuberty. For most previous in vitro studies immature rodent testes have been the source of Sertoli cells and these have shown little proliferative ability when cultured. We have isolated and characterized Sertoli cells from human cadaveric testes from seven donors ranging from 12 to 36 years of age. The cells proliferated readily in vitro under the optimized conditions used with a doubling time of approximately 4 days. Nuclear 5-ethynyl-2′-deoxyuridine (EdU) incorporation confirmed that dividing cells represented the majority of the population. Classical Sertoli cell ultrastructural features, lipid droplet accumulation, and immunoexpression of GATA-4, Sox9, and the FSH receptor (FSHr) were observed by electron and fluorescence microscopy, respectively. Flow cytometry revealed the expression of GATA-4 and Sox9 by more than 99% of the cells, and abundant expression of a number of markers indicative of multipotent mesenchymal cells. Low detection of endogenous alkaline phosphatase activity after passaging showed that few peritubular myoid cells were present. GATA-4 and SOX9 expression were confirmed by reverse transcription polymerase chain reaction (RT-PCR), along with expression of stem cell factor (SCF), glial cell line-derived neurotrophic factor (GDNF), and bone morphogenic protein 4 (BMP4). Tight junctions were formed by Sertoli cells plated on transwell inserts coated with fibronectin as revealed by increased transepithelial electrical resistance (TER) and polarized secretion of the immunoregulatory protein, galectin-1. These primary Sertoli cell populations could be expanded dramatically in vitro and could be cryopreserved. The results show that functional human Sertoli cells can be propagated in vitro from testicular cells isolated from adult testis. The proliferative human Sertoli cells should have important applications in studying infertility, reproductive toxicology, testicular cancer, and spermatogenesis, and due to their unique biological properties potentially could be useful in cell therapy.

Apoptosis resistance in HIV-1 persistently-infected cells is independent of active viral replication and involves modulation of the apoptotic mitochondrial pathway

BackgroundHIV triggers the decline of CD4+ T cells and leads to progressive dysfunction of cell-mediated immunity. Although an increased susceptibility to cell death occurs during the acute phase of HIV infection, persistently-infected macrophages and quiescent T-cells seem to be resistant to cell death, representing a potential reservoir for virus production.ResultsLymphoid (H9/HTLVIIIB and J1.1) and pro-monocytic (U1) HIV-1 persistently-infected cell lines were treated with hydrogen peroxide (H2O2) and staurosporine (STS) for 24 h, and susceptibility to apoptosis was evaluated and compared with uninfected counterparts (H9, Jurkat and U937 respectively). When exposed to different pro-apoptotic stimuli, all persistently-infected cell lines showed a dramatic reduction in the frequency of apoptotic cells in comparison with uninfected cells. This effect was independent of the magnitude of viral replication, since the induction of viral production in lymphoid or pro-monocytic cells by exposure to TNF-α or PMA did not significantly change their susceptibility to H2O2- or STS-induced cell death. A mechanistic analysis revealed significant diferences in mitochondrial membrane potential (MMP) and caspase-3 activation between uninfected and persistently-infected cells. In addition, Western blot assays showed a dramatic reduction of the levels of pro-apototic Bax in mitochondria of persistently-infected cells treated with H2O2 or STS, but not in uninfected cells.ConclusionThis study represents the first evidence showing that resistance to apoptosis in persistently-infected lymphoid and monocytic cells is independent of active viral production and involves modulation of the mitochondrial pathway. Understanding this effect is critical to specifically target the persistence of viral reservoirs, and provide insights for future therapeutic strategies in order to promote complete viral eradication.

Identification of galectins as novel regulators of platelet signaling and function

Platelet activation at sites of vascular injury leads to the formation of a hemostatic plug. Activation of platelets is therefore crucial for normal hemostasis. However, uncontrolled platelet activation may also lead to the formation of occlusive thrombi that can cause ischemic events. Platelets can be activated by soluble molecules including thrombin, TXA2, adenosine diphosphate (ADP), and serotonin or by adhesive extracellular matrix (ECM) proteins such as von Willebrand factor and collagen. In this article, we review recent advances on the role of galectins in platelet physiology. By acting in either soluble or immobilized form, these glycan‐binding proteins trigger platelet activation through modulation of discrete signaling pathways. We also offer new hypotheses and some speculations about the role of platelet–galectin interactions not only in hemostasis and thrombosis but also in inflammation and related diseases such as atherosclerosis and cancer.

Consensus guidelines for the use and interpretation of angiogenesis assays

AbstractThe formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

Abstract 465: Progestin-driven regulatory T cells directly promote an aggressive and metastatic phenotype in triple-negative breast cancer

The immune system plays key roles in the recognition and elimination of most tumors. Progesterone (Pg) can shape the immune response favoring a tolerogenic rather than a pro-inflammatory adaptive response. As hormone replacement therapies, supplementation and hormone-based contraceptives have been associated with an increase of malignant breast neoplasia we decided to investigate how progestins can regulate different key immune cell populations in the tumor microenvironment and their influence to tumor progression. To address this issue we used the highly metastatic, triple-negative 4T1 breast tumor. Balb/c mice treated with Pg or its synthetic analog, medroxyprogesterone acetate, showed an increased frequency of Foxp3+ regulatory T cells (Tregs) in draining lymph nodes (DLN) and an impaired antitumor response elicited by a decreased production of IL-17 and IFN-γ by CD4+ T cells (p Citation Format: Tomas Dalotto Moreno, Juan Pablo Cerliani, Diego Omar Croci, Santiago Patricio Mendez-Huergo, Florencia Moses, Gabriel Adrian Rabinovich, Mariana Salatino. Progestin-driven regulatory T cells directly promote an aggressive and metastatic phenotype in triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 465. doi:10.1158/1538-7445.AM2015-465

Abstract 1573: The progesterone analogue, Norgestrel, impairs tumor immunity and promotes metastatic breast cancer progression

The immune system is responsible for recognition and elimination of cancer cells. Tumors, however, can elicit different evasive mechanisms that contribute to immune escape Thus, understanding the immunosuppressive pathways that hamper tumor immunity is critical for the identification of novel therapeutic targets. Progestin-containing hormonal supplementation (as hormone replacement therapy or contraceptive) has been associated with an increase in the risk of breast cancer incidence and recurrence. Moreover, progesterone, as a natural hormone, generally impairs cellular immune responses responsible of preventingfetal rejection during pregnancy. We postulated that progestins and in particular the widely used contraceptive Norgestrel may impair tumor immunity and foster immunosuppressive tumor microenvironments. We injected Balb/c mice with the triple-negative 4T1breast tumor,, and treated them with a Norgestrel releasing depot for 30 days. Norgestrel-treated mice displayed a higher frequency of suppressive regulatory T cells (Tregs) in draining lymph nodes and tumors which displayed a CD4+CD25+Foxp3+CD44+ phenotype. Accordingly, norgestrel-treated mice showed greater number of exhausted PD1+ Tim-3+ CD8 T cells in the tumor microenvironment. Furthermore, in vitro exposure to Norgestrel also promoted differentiation and expansion of Tregs which activated the mTOR pathway assessed by phosphorylation of S6 ribonucleoprotein. These effects could not be prevented by the progesterone nuclear receptor antagonist, Zk 230211, suggesting that Norgestrel acts preferentially through membrane progesterone receptors. Although no significant difference regarding tumor growth could be observed, Norgestrel-treated mice showed greater number of lung metastasis. Mechanistically, Norgestrel-driven Treg cells showed increased expression of RANKL, a protein involved in breast cancer development and progression. Our results identify a novel progestin-mediated tumor-promoting mechanism and indicate caution onthe use of Norgestrel-supplementation therapies. Our findings suggest that Norgestrel may impair tumor immunity, thereby promoting metastatic dissemination of breast cancer cells. Citation Format: Tomas Dalotto Moreno, Gabriel Adrian Rabinovich, Mariana Salatino. The progesterone analogue, Norgestrel, impairs tumor immunity and promotes metastatic breast cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1573.

Abstract 400: Modulation of the metastatic microenvironment by soluble factors released by lung adenocarcinoma cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Soluble factors from primary tumors can modify target organs for metastasis through mobilization of bone marrow derived cells (BMDCs), which are recruited to tumors and to metastatic target organs and facilitate tumor growth and metastasis. Here we aimed to study early cellular and molecular events occurring in the lung, the metastatic target organ of the murine lung adenocarcinoma cell line LP07. We assessed by flow cytometry the numbers of BMDC positive for VEGFR1, SCF, CD34 or CXCR4 and the numbers of myeloid suppressor cells (CD11b+/Gr1+) and regulatory T cells (CD4+/CD25+/FOXP3+) in single cell lung suspensions obtained from 15-day tumor-bearing and control mice. Control and tumor-conditioned lungs of inoculated mice showed comparable amounts of these cell populations. To assess whether the primary tumor stimulates subsequent formation of macroscopic metastasis, mice bearing an intraperitoneal millipore chamber -that allows the diffusion of soluble molecules but not cell extravasation- loaded with LP07 cells or medium, were challenged with a LP07 intravenous inoculum at day 15. The presence of LP07 loaded chambers decreased the number of lung metastases three weeks after the inoculum compared to control mice (p≪0.05). In a different approach, mice were daily injected for 15 days with LP07 conditioned medium or control medium, and then challenged with a LP07 intravenous inoculum. When evaluated at day 21 post LP07 cell inoculation, the number of metastatic lung nodules was significantly lower (p≪0.05) in the group treated with LP07 conditioned medium. Besides, when we assessed matrix metalloproteinase (MMP) activity in lung homogenates from 15 day-tumor bearing and control mice, we found a significant increase in MMP-9 activity (p≪0.05) as assessed by semi-quantitative zymography in lungs from tumor-bearing mice.In conclusion, in the LP07 lung adenocarcinoma model, tumor cells secrete soluble factors that induce certain changes in the metastatic target organ, as the increase of MMP-9 activity. Moreover, in this experimental metastatic approach, a decrease in metastasis development was observed; the mechanisms underlying this phenomenon deserve further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 400. doi:10.1158/1538-7445.AM2011-400

Abstract 404: Murine lymphomas selected by growth rate: Aggressiveness, galectin-1 (Gal-1) expression and response to low doses of cyclophosphamide (Cy)

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC L-DGE is a spontaneous poorly differentiated murine lymphoma. During tumor growth it develops lymph node metastasis at a low frequency. Gal-1 is an immunomodulatory protein that acts as a regulator of T cell homeostasis and plays an important role in immune tolerance and tumor-immune escape. Overexpression of Gal-1 is associated with tumor progression and metastasis. Based on our previous findings, our aim was to obtain by a divergent selection two L-DGE variants that differ in tumor growth rate in order to study Gal-1 expression as well as their response to Cy. Two selection experiments were carried out. 1) L-DGE was s.c. implanted on 12 BALB/c mice (day 0). On day 14 the tumor displaying the largest volume was chosen as donor for the next passage to 4 mice. This higher-volume-selection was repeated for 25 serial passages, every 14 days and the tumor thus obtained was named L-DGE/M. 2) L-DGE was s.c. implanted on 12 BALB/c mice (day 0). On day 21, the tumor with the lowest growth rate was chosen as a donor and implanted into 6 mice and repeated through 16 serial passages. As a result of this selection L-DGE/L was obtained. We challenged mice (n=20/group) with L-DGE, L-DGE/M and L-DGE/L and studied tumor growth, metastatic capacity, galectin-1 expression and response to a single low dose Cy (25 mg/kg). Tumor volume vs. Time data was adjusted with an exponential curve and the growth rate was calculated. Gal-1 expression was studied by Western Blot in tumor and metastases homogenates. L-DGE/M growth rate was higher than that of L-DGE (p<0.01). L-DGE/L grew slower than L-DGE but did not differ statistically. The % of metastasis displayed by L-DGE/M was higher than that displayed by L-DGE (p=0.009) whereas L-DGE/L did not differ from L-DGE. Median survival times were 15, 20 and 26 days for L-DGE/M, L-DGE and L-DGE/L, respectively (p<0.0001). Tumor regressions were also different: 5%, 20% and 30%, respectively. Interestingly, Gal-1 expression in L-DGE/M primary tumors was higher (p=0.042) and in L-DGE/L lower (p=0.013) than in L-DGE, whereas in lymph-node metastases from L-DGE/M and L-DGE was 20% lower than their respective primary tumors, a pattern that we had already found in a rat lymphoma model. Finally, Cy treatment had different impact on tumor progression on these variants. While it increased median survival times of L-DGE, L-DGE/L and L-DGE/M-bearing mice, it induced exponential tumor volume decay with different rates on L-DGE, L-DGE/L and L-DGE/M (p=0.0001). Two lymphoma variants were obtained, of which, the more aggressive overexpresses Gal-1 in primary tumor while the slowly growing variant underexpresses this immunomodulatory protein. These differences also correlate with different responses to immunomodulatory doses of Cy. These results show a strong relationship between galectin-1 expression and tumor aggressiveness, a fact that could be of great value for prognosis and therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 404.