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Glioblastoma (GBM) is the most frequent malignant primary tumor of the CNS in adults, with a median survival of 14.6 months after diagnosis. The effectiveness of GBM therapies remains poor, highlighting the need for new therapeutic alternatives. In this work, we evaluated the effect of 4-methylumbelliferone (4MU), a coumarin derivative without adverse effects reported, in combination with temozolomide (TMZ) or vincristine (VCR) on U251, LN229, U251-TMZ resistant (U251-R) and LN229-TMZ resistant (LN229-R) human GBM cells. We determined cell proliferation by BrdU incorporation, migration through wound healing assay, metabolic and MMP activity by XTT and zymography assays, respectively, and cell death by PI staining and flow cytometry. 4MU sensitizes GBM cell lines to the effect of TMZ and VCR and inhibits metabolic activity and cell proliferation on U251-R cells. Interestingly, the lowest doses of TMZ enhance U251-R and LN229-R cell proliferation, while 4MU reverts this and even sensitizes both cell lines to TMZ and VCR effects. We showed a marked antitumor effect of 4MU on GBM cells alone and in combination with chemotherapy and proved, for the first time, the effect of 4MU on TMZ-resistant models, demonstrating that 4MU would be a potential therapeutic alternative for improving GBM therapy even on TMZ-refractory patients., (© 2023. The Author(s).)

Aims: Despite continuous improvement in the treatment of acute leukemia, new therapies are still needed to overcome resistance and reduce adverse effects. The aim of this work was to study the tumor-suppressive effects of 4-methylumbelliferone (4MU) in human acute leukemia cell lines. In addition, we aimed to address the extent of these effects in relation to the inhibition of hyaluronic acid (HA) synthesis., Main Methods: HA levels were measured by an ELISA-like assay. Human acute leukemia cell lines were treated with 4MU, HA or their combination. Cell proliferation was assessed by the [ 3 H]-Tdr uptake assay, metabolic activity by the XTT assay and cell death was determined by DAPI, AO/EB and AnnexinV-PE/7-AAD staining. Senescence induction was evaluated by SA-β-Gal and C12FDG staining. Total and surface RHAMM expression levels were assessed by flow cytometry and fluorescence microscopy., Key Findings: 4MU reduced metabolic activity and inhibited cell proliferation in all leukemia cells, and these effects were explained by the induction of senescence or cell death depending on the cell line evaluated. Exogenous HA failed to prevent most of the tumor-suppressive effects observed. Results from this work suggest that the tumor-suppressive effects exerted by 4MU would be explained by HA-synthesis-independent mechanisms., Significance: These findings broaden the knowledge of 4MU as a potential treatment in acute leukemia. We report for the first time the existence of tumor-suppressive effects of 4MU on human acute leukemia cell lines that are independent of its role as HA-synthesis inhibitor., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The extracellular matrix plays a key role in cancer progression. Hyaluronan, the main glycosaminoglycan of the extracellular matrix, has been related to several tumor processes. Hyaluronan acts through the interaction with cell membrane receptors as CD44 and RHAMM and triggers signaling pathways as MEK/ERK. 4-methylumbelliferone (4MU), a well-known hyaluronan synthesis inhibitor, is a promising alternative for cancer therapy. 4MU is a coumarin derivative without adverse effects that has been studied in several tumors. However, little is known about its use in glioblastoma (GBM), the most malignant primary brain tumor in adults. Glioblastoma is characterized by fast growth, migration and tissue invasiveness, and a poor median survival of the patients after treatment. Several reports linked glioblastoma progression with HA levels and even with CD44 and RHAMM expression, as well as MEK/ERK activation. Previously, we showed on a murine GBM cell line that HA enhances GBM migration, while 4MU markedly inhibits it. In this work we showed for the first time, that 4MU decreases cell migration and induces senescence in U251 and LN229 human GBM cell lines. Furthermore, we observed that HA promotes GBM cell migration on both cell lines and that such effects depend on CD44 and RHAMM, as well as MEK/ERK signaling pathway. Interestingly, we observed that the exogenous HA failed to counteract the effects of 4MU, indicating that 4MU effects are independent of HA synthesis inhibition. We found that 4MU decreases total CD44 and RHAMM membrane expression, which could explain the effect of 4MU on cell migration. Furthermore, we observed that 4MU increases the levels of RHAMM inside the cell while decreases the nucleus/cytoplasm relation of p-ERK, associated with 4MU effects on cell proliferation and senescence induction. Overall, 4MU should be considered as a promising therapeutic alternative to improve the outcome of patients with GBM., (© 2021. The Author(s).)

Glioblastoma (GBM), the most frequent primary tumor of the central nervous system, has a median survival of 14.6 months. 4-Methylumbelliferone (4MU) is a coumarin derivative widely used as a hyaluronan synthesis inhibitor with proven antitumor activity and without toxic effects reported. We aim to evaluate the antitumor effect of 4MU alone or combined with temozolomide (TMZ) on a GBM cell line, its absence of toxicity on brain cells and its selectivity for tumor cells. The antitumor effect of 4MU alone or combined with TMZ was evaluated on GL26 cells by assessing the metabolic activity through the XTT assay, cell proliferation by BrdU incorporation assay, migration by the wound healing assay, cell death by fluorescein diacetate/propidium iodide (FDA/PI) staining, apoptosis by membrane asymmetry and DNA fragmentation and metalloproteinase activity by zymography. The levels of hyaluronan and its capacity to counteract the effects of 4MU and the expression of RHAMM and CD44 were also determined. The toxicity and selectivity of 4MU were determined by XTT assay and PI staining on normal brain primary cell culture (NBPC-GFP) and GL26/NBPC-GFP cocultures. The GL26 cells expressed RHAMM but not CD44 while synthetized hyaluronan. 4MU decreased hyaluronan synthesis, diminished proliferation and induced apoptosis while reducing cell migration and the activity of metalloproteinases, which was restored by addition of hyaluronic acid. Furthermore, 4MU sensitized GL26 cells to the TMZ effect and showed selective toxicity on tumor cells without exhibiting neurotoxic effects. We demonstrated for the first time the cytotoxic effect of 4MU on GBM cells, highlighting its potential usefulness to improve GBM treatment., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Advances in cancer biology are revealing the importance of the cancer cell microenvironment on tumorigenesis and cancer progression. Hyaluronan (HA), the main glycosaminoglycan in the extracellular matrix, has been associated with the progression of glioblastoma (GBM), the most frequent and lethal primary tumor in the central nervous system, for several decades. However, the mechanisms by which HA impacts GBM properties and processes have been difficult to elucidate. In this review, we provide a comprehensive assessment of the current knowledge on HA's effects on GBM biology, introducing its primary receptors CD44 and RHAMM and the plethora of relevant downstream signaling pathways that can scramble efforts to directly link HA activity to biological outcomes. We consider the complexities of studying an extracellular polymer and the different strategies used to try to capture its function, including 2D and 3D in vitro studies, patient samples, and in vivo models. Given that HA affects not only migration and invasion, but also cell proliferation, adherence, and chemoresistance, we highlight the potential role of HA as a therapeutic target. Finally, we review the different existing approaches to diminish its protumor effects, such as the use of 4-methylumbelliferone, HA oligomers, and hyaluronidases and encourage further research along these lines in order to improve the survival and quality of life of GBM patients., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Hyaluronan (HA) is the major glycosaminoglycan present in the extracellular matrix. It is produced by some tumours and promotes proliferation, differentiation and migration among others cellular processes. Gestational trophoblastic disease (GTD) is composed by non-tumour entities, such as hydatidiform mole (HM), which is the most common type of GTD and also malignant entities such as choriocarcinoma (CC) and placental site trophoblastic tumour (PSTT), being CC the most aggressive tumour. Although there is a growing understanding of GTD biology, the role of HA in the pathogenesis of this group of diseases remains largely unknown. The aim of this work was to study the role of HA in the pathogenesis of GTD by defining the expression pattern of HA and its receptors CD44 and RHAMM, as well as to determine if HA can modulate proliferation, differentiation and migration of CC cells. Receptors and signalling pathways involved were also analyzed. We demonstrated that HA and RHAMM are differently expressed among GTD entities and even among trophoblast subtypes. We also showed that HA is able to enhance the expression of extravillous trophoblast markers and also to induce migration of JEG-3 cells, the latter mediated by RHAMM as well as PI3K and MAPK pathways. These findings indicate a novel regulatory mechanism for CC cell biology and also contribute to the understanding of GTD pathophysiology.

Chronic myeloid leukemia (CML) is a myeloproliferative syndrome characterized by the presence of the Philadelphia chromosome which encodes a constitutively activated tyrosine kinase (BCR-ABL). The first line treatment for CML consists on BCR-ABL inhibitors such as Imatinib. Nevertheless, such treatment may lead to the selection of resistant cells. Therefore, it is of great value to find molecules that enhance the anti-proliferative effect of first-line drugs. Hyaluronan is the main glycosaminglican of the extracellular matrix which is involved in tumor progression and multidrug resistance. We have previously demonstrated that the inhibition of hyaluronan synthesis by 4-methylumbelliferone (4MU) induces senescence and can revert Vincristine resistance in CML cell lines. However, the effect of 4MU on Imatinib therapy remains unknown. The aim of this work was to determine whether the combination of 4MU with Imatinib is able to modulate the proliferation as well as apoptosis and senescence induction in human CML cell lines. For this purpose the ATCC cell line K562, and its multidrug resistant derivate, Kv562 were used. Cells were exposed to 4MU, Imatinib or a combination of both. We demonstrated that 4MU and Imatinib co-treatment abrogated the proliferation of both cell lines. However, such co-treatment did not increase the levels of apoptosis when compared with the treatment with Imatinib alone. For both cell lines the mechanisms of tumor suppression involved was senescence, since the combination of 4MU and Imatinib arrested the cell cycle and increased senescence associated β-galactosidase activity and senescence associated heterochromatin foci presence when compared to each drug alone. Moreover, 4MU, Imatinib and 4MU + Imatinib decreased pAkt/Akt ratio in both cell lines and reduced the pERK/ERK ratio only in K562 cells. These findings highlight the potential use of 4MU together with Imatinib for CML therapy.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of ruminant paratuberculosis. The aim of this study was to evaluate the biological behavior of different Argentinean strains of MAP in two bovine infection models: macrophage (in vitro) and calf (in vivo) through the evaluation of early immune responses at the peripheral and local levels. Two MAP strains (A and C) were selected taking into account the different patterns of TNF-α and IL-10 secretion displayed by infected bovine macrophages in vitro. Two groups of calves were infected with 250mg of total wet weight live MAP: strain A infected group (MA, n=3), strain C infected group (MC, n=2). Another group of animals was mock-infected (MI, n=3). Infection was confirmed by MAP culture of feces and microscopic observation of granulomatous lesions in the gut tissue. All infected calves showed positive results in the DTH skin test. A significant increase in peripheral CD4CD25(+) cells in MC group on day 150 was detected. The specific cellular immune response developed allowed the identification of the infection as early as 30days in the MA group. However, the percentage of CD8CD25(+) cells was significantly increased on day 120 in MC group. Significant differences between groups in proliferation and cellular responses were also detected in ileocecal lymph node samples. In summary, the strains of MAP employed herein induced differential immune responses in peripheral cells, in the proliferative responses and in cell functionality at the local level. Our findings support the hypotheses that the in vitro behavior displayed by macrophages could be a tool to identify differences among MAP strains infecting bovines and that the host-pathogen interactions occurring upon infection are dependent on the strain of MAP involved., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Chronic myeloid leukemia is a myeloproliferative syndrome characterized by the presence of the Philadelphia chromosome (Ph), generated by a reciprocal translocation occurring between chromosomes 9 and 22 [t(9;22)(q34;q11)]. As a consequence, a fusion gene (bcr-abl) encoding a constitutively active kinase is generated. The first-line treatment consists on BCR-ABL inhibitors such as Imatinib, Nilotinib and Dasatinib. Nevertheless, such treatment may lead to the selection of resistant cells. Therefore, finding molecules that enhance the anti-proliferative effect of first-line drugs is of value. Hyaluronan oligomers (oHA) are known to be able to sensitize several tumor cells to chemotherapy. We have previously demonstrated that oHA can revert Vincristine resistance in mouse lymphoma and human leukemia cell lines. However, little is known about the role of oHA in hematological malignancies. The aim of this work was to determine whether oHA are able to modulate the anti-proliferative effect of Imatinib in chronic myeloid leukemia (CML) cell lines. The effect on apoptosis and senescence as well as the involvement of signaling pathways were also evaluated. For this purpose, the human CML cell lines K562 and Kv562 (resistant) were used. We demonstrated that oHA sensitized both cell lines to the anti-proliferative effect of Imatinib increasing apoptosis and senescence. Moreover, this effect would be accomplished through the down-regulation of the PI3K signaling pathway. These findings highlight the potential of oHA when used as a co-adjuvant therapy for chronic myeloid leukemia., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic granulomatous enteritis in ruminants. Understanding the protective immune response following infection is crucial to improve the diagnosis and the development of vaccines against this disease. The goal of this work was to assess whether specific antibodies were able to modulate the macrophage response to MAP infection by evaluating apoptosis and TNF-α secretion in an in vitro model. Sera from healthy (n=2), MAP-infected (n=3) and lipoarabinomannan (LAM)-immunized (n=3) bovines were evaluated. LAM was chosen as immunogen due to its relevant role in mycobacterial pathogenesis. We demonstrated by two different techniques (Acridine Orange/Ethidium Bromide microscopy and Annexin V/7-Amino-Actinomycin D flow cytometry) that the immune sera from both, MAP-infected and LAM-immunized bovines, significantly increased macrophage apoptosis in infected cultures. Comparable levels of apoptosis were detected when MAP was pre-incubated with purified specific antibodies instead of whole serum. Furthermore, this effect was accompanied by a significantly higher secretion of TNF-α. These results strongly suggest that specific antibodies could limit the impact of MAP on the apoptosis of bovine cells. This work would contribute to elucidate the role of the specific antibody response in bovine JD and its prevention., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Context: Therapy for snakebites relies on the application of antivenoms, which may be produced with different immunogenic mixtures of venom and possess different pharmaceutical characteristics. For these reasons, immunological cross-reactivity and heterologous neutralization were analyzed relative to the protein content of three antivenoms used in the Americas., Methods: The antivenoms studied were composed of equine F(ab')2 fragments from animals immunized with Crotalinae venoms. The antivenoms were tested against venoms of seven pit viper species from Argentina, seven from Mexico, one from Costa Rica, and one from Colombia., Results: Immunoblotting showed high cross-reactivity of all major protein bands with all the antivenoms tested. ELISA results also showed high cross-reactivity among the different venoms and antivenoms, and a high heterologous neutralization was observed. The results can be interpreted in different ways depending on whether the reactivity is considered in terms of the volume of antivenom used or by the amount of protein contained in this volume of antivenom. The antivenoms with high immunochemical reactivity and neutralizing capacity were those with higher protein content per vial; but when doses were adjusted by protein content, antivenoms of apparently lower neutralizing capacity and immunochemical reactivity showed at least similar potency and reactivity although volumetrically at higher doses., Conclusion: Protein content relative to neutralization potency of different products must be taken into account when antivenoms are compared, in addition to the volume required for therapeutic effect. These results show the importance of obtaining high-affinity and high-avidity antibodies to achieve good neutralization using low protein concentration and low-volume antivenoms.

Hyaluronan (HA) is one of the major components of the extracellular matrix. Several solid tumors produce high levels of HA, which promotes survival and multidrug resistance (MDR). HA oligomers (oHAs) can block HA effects. However, little is known about the role of HA in hematological malignancies. The aim of this work was to determine whether HA or its oligomers can modulate the proliferation of leukemia cells as well as their effect on MDR. Receptors and signaling pathways involved were also analyzed. For this purpose, the human leukemic cell lines K562 and Kv562, which are sensitive and resistant to Vincristine (VCR), respectively, were used. We demonstrated that HA induced cell proliferation in both cell lines. On K562 cells, this effect was mediated by cluster differentiation 44 (CD44) and activation of both phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways, whereas on Kv562 cells, the effect was mediated by receptor for hyaluronan-mediated motility (RHAMM) and PI3K/Akt activation. The inhibition of HA synthesis by 4-methylumbelliferone (4MU) decreased cell line proliferation and sensitized Kv562 to the effect of VCR through P-glycoprotein (Pgp) inhibition, in both cases with senescence induction. Moreover, oHAs inhibited K562 proliferation mediated by CD44 as well as Akt and ERK down-regulation. Furthermore, oHAs sensitized Kv562 cells to VCR by Pgp inhibition inducing senescence. We postulate that the synthesis of HA would promote leukemia progression mediated by the triggering of the above-mentioned proliferative signals. These findings highlight the potential use of oHAs and 4MU as coadjuvant for drug-resistant leukemia.

A phospholipase enzyme was separated by chromatography from the venom of the snake Bothrops (Rhinocerophis) ammodytoides and characterized. The experimentally determined molecular weight was 13,853.65 Da, and the full primary structure was determined by Edman degradation and mass spectrometry analysis. The enzyme contains 122 amino acids residues closely stabilized by 7 disulfide bridges with an isoelectric point of 6.13. Sequence comparison with other known secretory PLA2 shows that the enzyme isolated belongs to the group II, presenting an aspartic acid residue at position 48 (numbered by convention as Asp49) of the active site, and accordingly displaying enzymatic activity. The enzyme corresponds to 3% of the total mass of the venom. The enzyme is mildly toxic to mice. The intravenous LD₅₀ of this phospholipase in CD-1 mice was around 6 μg/g of mouse body weight (more exactly 117 μg/mouse of 20 g) and the minimal mortal dose (MMD) was estimated to be close to 10 μg/g. In contrast, the LD₅₀ of the venom was circa 2 μg/g mouse body weight. Toxicological analyses of the purified enzyme were performed in vitro and in vivo using experimental animals (mice and rats). The enzyme at high doses caused pulmonary congestion, intraperitoneal bleeding, inhibition of clot retraction and muscle tissue alterations with increasing of creatine kinase levels., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objective: To analyze the effect of corticosteroid administration on the concentration of hyaluronan (HA) in bronchoalveolar lavage (BAL) in a murine model of eosinophilic airway inflammation and to study the mechanisms involved., Materials and Methods: Untreated-mice or mice treated with 1 μg/g/day betamethasone (Bm) or 0.25 μg/g/day(-1) budesonide (Bd) were sensitized and challenged with Dermatophagoides pteronyssinus (Dp) or saline (control group). The concentration of HA in BAL was determined by ELISA. In vitro migration assays were performed using a Boyden chamber and the expression of HA synthases (HAS) was analyzed by RT-PCR., Results: We found a significant increase (P < 0.01) in the levels of HA in BAL from Dp-treated mice that was prevented by Bm or Bd. Corticosteroids also inhibited the increase in HAS expression, and the phosphorylation of Akt and ERK in the lungs of challenged mice. Finally, we found that low molecular weight HA induces the chemotaxis of BAL cells in vitro through a mechanism mediated by CD44., Conclusion: We conclude that corticosteroids prevent the increase in HA in BAL from Dp-challenged mice. This effect is associated with reduced expression of HAS and reduced phosphorylation of Akt and ERK in the lungs of challenged mice.

We report a study of toxic and enzymatic activities of Bothrops neuwiedi complex venoms collected from specimens of different regions of Argentina and a pool of these same venoms. Were determined lethal, hemorrhagic and pro-coagulant (plasma and fibrinogen) doses and the neutralization of these activities by a bivalent antivenom. The electrophoretic pattern of different regions venom was studied by SDS-PAGE. All samples exhibited lethal potencies, hemorrhagic and coagulant (plasma and fibrinogen) activities with potencies concordant with previous studies. The only conspicuous difference in the toxicological pattern of Bothrops diporus venoms was the low-thrombin-like activity found in one sample. The antivenom used in this study could neutralize all the toxic activities tested and the neutralizing potency of the antivenom was comparable for all samples. Despite the wide distribution of B. neuwiedi complex throughout Argentina and the evident morphological variation between B. diporus (B. neuwiedi complex), this study establishes a remarkably similar toxicity profile throughout its range. This is the first systematic study on the regional variation of enzymatic and toxic activities of venom from species belonging to the B. neuwiedi complex, one of the snakes of highest sanitary importance in South America and their neutralization by the type of antivenom most commonly used in the South of South America., (Copyright © 2011. Published by Elsevier Ltd.)

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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

HLA-G is a nonclassical MHC class I antigen that displays tolerogenic functions; MICA is a stress-regulated molecule recognized by NKG2D cytotoxicity-activating receptor expressed by NK and T cells subsets. We evaluated HLA-G isoforms and MICA mRNA levels in peripheral blood mononuclear cells (PBMCs) and in biopsies from kidney allograft recipients with acute rejection (AR), chronic rejection (CR), and stable graft evolution (SE). HLA-G1 was the only transcript resulted from amplification, both in PBMCs as in biopsy samples. HLA-G1 mRNA levels in PBMCs from 9/10 patients with CR, 7/9 with AR and 8/10 healthy volunteers were below the median value of SE patients. The analysis of biopsies revealed that patients with AR (n=6), who overcame rejection had a tendency towards higher HLA-G1 levels than those with nephrotoxic acute tubular necrosis (ATN) (n=3). Similar levels of MICA expression were observed in PBMCs from AR, CR, SE and C groups; MICA expression levels were similar also in biopsy specimens from AR and nephrotoxic ATN patients. No correlation was found between MICA expression and the graft state. These preliminary results suggest that HLA-G1 isoforms, but not MICA mRNA levels, may provide a marker for measuring the state of kidney allograft, and be the basis for further studies that may establish the influence of these molecules in renal allograft rejection or acceptance.

Chemotherapy aims to limit proliferation and induce apoptotic cell death in tumor cells. Owing to blockade of signaling pathways involved in cell survival and proliferation, nuclear factor kappaB (NF-kappaB) inhibitors can induce apoptosis in a number of hematological malignancies. The efficacy of conventional chemotherapeutic drugs, such as vincristine (VCR) and doxorubicine (DOX), may be enhanced with combined therapy based on NF-kappaB modulation. In this study, we evaluated the effect of caffeic acid phenylethyl ester (CAPE) and MG-132, two nonspecific NF-kappaB inhibitors, and conventional chemotherapeutics drugs DOX and VCR on cell proliferation and apoptosis induction on a lymphoblastoid B-cell line, PL104, established and characterized in our laboratory. CAPE and MG-132 treatment showed a strong antiproliferative effect accompanied by clear cell cycle deregulation and apoptosis induction. Doxorubicine and VCR showed antiproliferative effects similar to those of CAPE and MG-132, although the latter drugs showed an apoptotic rate two-fold higher than DOX and VCR. None of the four compounds showed cytotoxic effect on peripheral mononuclear cells from healthy volunteers. CAPE- and MG-132-treated bone marrow cells from patients with myeloid and lymphoid leukemias showed 69% (P < .001) and 25% decrease (P < .01) in cell proliferation and 42% and 34% (P < .01) apoptosis induction, respectively. Overall, our results indicate that CAPE and MG-132 had a strong and selective apoptotic effect on tumor cells that may be useful in future treatment of hematological neoplasias.

Upregulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway has been described in some tumors related to multidrug resistance (MDR). The aim of this work was to analyze the relationship between PI3K/Akt, MDR and NF-kappaB in murine lymphoma cell lines resistant to vincristine (LBR-V160) and doxorubicin (LBR-D160) as well as in the sensitive line (LBR-). PI3K/Akt activity, analyzed by phosphatidylinositol trisphosphate production and phosphorylated Akt (p-Akt) expression, was higher in the resistant cell lines than in the sensitive one and inhibition with wortmannin or LY294002 improved apoptosis in the resistant cell lines. Vincristine but not doxorubicin increased p-Akt expression whereas co-treatment with PI3K inhibitors and vincristine increased apoptosis in the three cell lines. Wortmannin and LY294002 inhibited P-glycoprotein (Pgp) function and also increased NF-kappaB activity. We concluded that the PI3K/Akt pathway is involved in MDR in lymphoma cell lines and PI3K/Akt inhibition correlates down-regulation of NF-kappaB activity and inhibition Pgp function.

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., ((c) 2007 Wiley-Liss, Inc.)

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a chronic granulomatous enteric disease in cattle. Among molecular components of Map, protein p34 was identified as specific and immunodominant for bovine B cells. In order to determine if specific antibodies could influence the course of Map pathogenesis, the interaction between bacteria and bovine macrophages was studied. Bovine polyclonal antibodies from 3 calves vaccinated with protein p34-cx, 6 calves vaccinated with heat-killed Map, 8 naturally infected, and 3 healthy calves -as negative controls- were used. Specific anti-Map, -p34-cx and -PPA-3 antibodies were evaluated and isotype characterized. Infected and Map vaccinated animals showed similar IgG1 and IgG2 response against Map whole bacteria. When p34-cx was used as the antigen, mainly IgG1 and IgG3 were detected in infected and only IgG1 in p34-cx vaccinated animals. Bovine polyclonal antibodies from three animals of each category were isolated and affinity purified through Map and p34-cx columns. The effect of these antibodies in association with Map and a transformed bovine peritoneal macrophage's cell line (Bov-Mac) as well as activation of NF-kappaB transcription factor was studied. Our results show that association of Map significantly increased in vitro after pretreatment with bovine anti-Map or anti-p34-cx antibodies obtained from vaccinated or infected cattle when compared with those of controls. Improved activation of NF-kappaB was detected in macrophages that ingested Map opsonized with either anti-Map or anti-p34-cx specific antibodies of infected or vaccinated calves, suggesting that both anti-Map and IgG1 anti-p34-cx antibodies support Map-macrophage interactions.

The materno-fetal interface has for long been considered as an immune privileged biological site and thus understanding the mechanisms underlying fetal survival have been the focus of intense research. In adults, survivin and Stat-3 proteins are involved in tolerance as well as the induction of apoptosis. However, the role of these molecules in pregnancy and development has not been addressed. We have evaluated the expression of survivin and Stat-3 in allogeneic mouse models of low abortions (CBA/J x Balb/c), abortion prone (CBA/J x DBA/2J) and stress-triggered abortions from DBA/2J-mated CBA/J mice. We show that survivin is over-expressed in abortion-prone mating on gestation day 7.5. This effect was also found in stress-exposed mice, whereas expression was low in normal pregnancy mice. The phosphorylated Stat-3 (p-Stat-3) was down regulated in high abortion mating compared with low abortion mating, CBA/J x Balb/c. The level of apoptosis was similar in the three groups studied. Our results suggest that high expression of survivin and low expression of p-Stat-3 are involved in pregnancy loss in mice.

Several studies indicate that hyaluronan oligosaccharides (oHA) are able to modulate growth and cell survival in solid tumors; however, no studies have been undertaken to analyze the effect of oHA on T-lymphoid disorders. In this work we showed that oHA were able to induce apoptosis in lymphoma cell lines. Since PI3-K/Akt and nuclear factor-kappaB (NF-kappaB) are major factors involved in cell survival and anti-apoptotic pathways in lymphoma cells, we hypothesized that oHA could induce apoptosis through inhibition of these pathways. oHA were identified by a method which allows characterization of length using a high pH anion exchange chromatography with pulse amperometric detection (HPAEC-PAD). oHA inhibited PIP(3) production (principal product of PI3-K activity) and reduced Akt phosphorylation levels, similarly to the specific inhibitor wortmannin. However, treatment with either oHA or wortmannin failed to inhibit constitutive NF-kappaB activity and modulate IkappaBalpha protein levels, suggesting that PI3-K and NF-kappaB signaling pathways are not related in the cell lines used. Cell behavior differed using native hyaluronan (HA), which induced PIP(3) production, Akt phosphorylation, and NF-kappaB activation, although not related with cell survival since treatment with native HA showed no effect on apoptosis. Our results suggest that oHA induce apoptosis by suppression of PI3-K/Akt cell survival pathway without involving NF-kappaB activation, through a mechanism that differs from the one mediated by native HA.

Multidrug resistance (MDR) is the main reason for failure of cancer therapy with resistance to apoptosis being one of the mechanisms involved. Constitutive NF-kappaB activity has been detected in many tumors contributing to oncogenesis and tumor survival whereas inhibition of NF-kappaB activity has proved to enhance cell death induced by chemotherapeutic agents. Consequently, the use of BAY 11-7082, an irreversible inhibitor of IkappaB-alpha phosphorylation, could be beneficial in the treatment of certain tumors. Although there are several reports which demonstrate a transient activation of NF-kappaB by cytotoxic drugs, little is known about the role of NF-kappaB activation in the development of a chemoresistant phenotype in leukemic cells. In this study, we analyzed the relationship between NF-kappaB and the survival of murine leukemic drug resistant cell lines. The modulation of this transcription factor by BAY 11-7082 and the chemotherapeutic agents vincristine and doxorubicin was evaluated. The effect of BAY 11-7082 on the expression of genes containing NF-kappaB-binding sites was also studied. We found that the cell lines LBR-V160 and LBR-D160 (resistant to vincristine and doxorubicin, respectively) presented higher constitutive NF-kappaB activity than the sensitive LBR- and the active complex contained both p50 and p65 subunits. BAY 11-7082 (3.5 microM) inhibited constitutive NF-kappaB activity in the three cell lines whereas the anticancer agents did not. Treatment with BAY 11-7082 induced a higher percentage of apoptosis in LBR-V160 and LBR-D160 than in LBR-. Cells treated with BAY 11-7082 displayed modulation of NF-kappaB-inducible genes such as IL-10, IL-15, TNF-alpha and TGF-beta. Taken together, these data suggest that suppression of constitutive NF-kappaB activity by BAY 11-7082 may be a useful treatment for MDR leukemias.

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.

Objective: To evaluate the effects of anti-CD44 IM7.8.1 antibody, HMW-HA and LMW-HA on leukocyte migration and adhesion, and the induction of proinflammatory mediators, in mouse air-pouch inflammation induced by zymosan., Methods: Leukocytes were obtained from zymosan-air pouches after the intra-pouch injection of anti-CD44 IM7.8.1, isotype control, HMW-HA, LMW-HA or PBS. TNF-alpha, IL-1beta and iNOS mRNA were estimated in leukocytes by semi-quantitative RT-PCR. Matrix metalloproteinases (MMPs) from exudates were evaluated by zymography and Western Blot. Adhesion and migration of leukocytes were evaluated in HA-coated plates and Boyden chambers respectively., Results: IM7.8.1 decreased iNOS mRNA levels and the activity of both MMP-9 and MMP-2 eight h after injection into zymosan air pouch while IM7.8.1, HMW-HA and LMW-HA had no effect on IL1-beta or TNF-alpha mRNA levels. Leukocytes from air pouch adhered to and migrated in vitro against both HMW-HA and LMW-HA. LMW-HA increased the number of leukocytes in the air pouch and iNOS mRNA levels as compared to PBS injection. In contrast, HMW-HA decreased leukocyte count and reduced iNOS mRNA levels. Paradoxically, the activity of both MMP-9 and MMP-2 was increased by HMW-HA and decreased by LMW-HA., Conclusions: Both CD44 and HA can modulate leukocyte migration and induction of proinflammatory mediators in mouse zymosan air pouch inflammation. IM7.8.1 had consistent anti-inflammatory effects, reducing iNOS, MMP-9 and MMP-2. HMW-HA and LMW-HA were able to modulate both the induction of proinflammatory mediators and leukocyte count in the air pouch.

Phoradendron liga (Gill. ex H. et A.) Eichl. is a Viscaceae widely distributed in Argentina. It has been commonly used in folk medicine as a substitute of the European mistletoe (Viscum album L.) to decrease high blood pressure due to their external similarity. In this study, the anatomical features as well as micromolecular and macromolecular analysis of this species are reported. Anatomical study has shown that Phoradendron liga presents as anatomic features: papillous cuticle, clusters in leaves and stems, and isodiametric stone cells only in stems. The analysis of flavonoids showed that this species produces C-glycosylflavones and 3-desoxyproanthocyanidins. Protein study showed a protein pattern with components ranging from 14 to 90 kDa and the presence of related epitopes between the species was demonstrated by cross recognition using anti-Phoradendron and anti-Viscum antisera of both species by Western blot assay. In addition, a galactose specific lectin (L-Phl) was isolated form Phoradendron liga extracts. These results are part of a comprehensive project on Argentine hemiparasite species destinated to be applied to quality control of commercial samples and disclosed their potential use as a potential source for immunomodulatory compounds.

Previous findings from our laboratory demonstrated that when used at low concentration (0.1 microg ml(-1)), CsA as well as its analog PSC 833 were able to revert the MDR phenotype, while at high concentration (1 microg ml(-1)) were able to induce apoptosis. CsA induced apoptosis in leukemia cell lines sensitive (LBR-) and resistant to vincristine (LBR-V160), and doxorubicin (LBR-D160), while PSC 833 only induced apoptosis in vincristine-resistant cell line (LBR-V160). In this work, we investigated mitochondrial-associated mechanisms during CsA- and PSC 833-induced apoptosis. Mitochondrial function was evaluated by recording changes in its transmembrane potential, cytochrome c release, and caspase activation cascade. Results showed that CsA- and PSC 833-induced apoptosis was associated with mitochondrial depolarization, through potentiometric measurements with JC-1 and DiOC(6) probes. Collapse of mitochondrial potential in these cell lines after CsA treatment was followed by cytochrome c release to the cytosol, reaching an increase of 2.61-fold in LBR-, 1.98-fold in LBR-V160, and 3.01-fold in the case of LBR-D160. However, in the case of PSC 833 treatment, induction of apoptosis in LBR-V160 was associated with mitochondrial depolarization followed by a lower cytochrome c release of 1.15-fold as compared with untreated cells. Caspase 3 activation was clearly observed in LBR-, LBR-V160, and LBR-D160 after CsA treatment, while in LBR-V160, PSC 833 was less effective inducing activation of this caspase. Neither caspase 6 nor 8 activity was observed in these three cell lines. Our results suggest that during CsA- and PSC 833-induced apoptosis, mitochondrial dysfunction occurs. This is mediated through mitochondrial events, associated with an evident decrease in DeltaPsi(m), cytochrome c release and caspase 3 activation.

Among the mechanisms that induce multidrug resistance (MDR), one of those most frequent is over-expression of a phosphoglycoprotein (Pgp) encoded in the mouse by the mdr-1 and mdr-3 genes. We have demonstrated that cyclosporin-A (CsA) as well as its analogue PSC 833 were able to revert the MDR phenotype in murine cell lines resistant to vincristine (LBR-V160) or doxorubicin (LBR-D160). The aim of this work was to evaluate the ability of PSC 833 and CsA to modulate mdr-1, mdr-3 and mrp-1 genes as well as to induce apoptosis analyzing the mechanism involved in the above tumor cell lines. By semi-quantitative RT-PCR, we demonstrated that mdr-3 was over-expressed in both resistant lines while mdr-1 was over-expressed only in LBR-V160; in contrast, mrp-1 expression was not evidenced in any of the cell lines. After treatment with 0.1 microg ml(-1) of either PSC 833 or CsA, LBR-V160 showed no changes in mdr-1 but decreased mdr-3 expression, while LBR-D160 failed to display any modification in the expression of these genes. Apoptosis was evidenced by fluorescence microscopy, S minuscule accumulation and agarose gel electrophoresis. Our results demonstrated that CsA (1 microg ml(-1)) was able to induce apoptosis in all cell lines: 18.31% (+/-4.46) for LBR-, 25.96% (+/-5.24) for LBR-V160 and 27.36% (+/-4.12) for LBR-D160, while PSC 833 (1 microg ml(-1)) only induced apoptosis 21.51% (+/-5.73) in LBR-V160 cell line. The expression of Bcl-2 family proteins (Bcl-2, Bax and Bcl-x(L)) was analyzed by flow cytometry showing high expression of the three proteins which was not significantly modified after treatment with either PSC 833 or CsA on the sensitive as well as on the resistant cell lines. Single stranded conformation polymorphisms analysis of p53 (Trp53) gene in the cell lines showed no mutation in exons 5-8 of the tumor suppressor gene. We conclude that depending on the concentration used, PSC 833 and CsA may act either by modulating the mdr-3 gene (0.1 microg ml(-1)) or by direct impact on the cells through induction of apoptosis (1 microg ml(-1)), in the latter case through a mechanism that might act independent of the Bcl-2 family proteins.

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 ofneoplastic 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.

Multidrug resistance (MDR) lines from a murine T-cell lymphoid leukemia were selected in increasing vincristine (VCR) or doxorubicin (DOX) concentrations. Surface markers were determined by flow cytometry in both resistant (LBR-V 160 and LBR-D 160) and sensitive (LBR-) cell lines. Results obtained revealed similar expression of CD25, CD24, CD8, CD4, C18 and CD44, while differences in binding to hyaluronic acid (HA) were found. LBR- and LBR-D 160 bound to HA only after phorbol ester (PMA) activation, while LBR-V160 failed to bind HA even after PMA treatment. Histopathological analysis disclosed that LBR-V160 was less invasive than LBR- and LBR-D160 cell lines. In vitro growth of cell lines analyzed by sulforhodamine-B uptake showed that doubling time for the three lines was 10.24 h (LBR-), 16.75 h (LBR-V160) and 16.29 h (LBR-D160). Mortality rate was determined after i.p. injection of 10(4) cells. Mice inoculated with LBR- died at 23 2.11) days, while those inoculated with LBR-V160 or LBR-D160 died at 41 (+/- 9.53) or 41 (+/- 4.96) days, respectively. Our results demonstrated that leukemic murine T cells cultured in the long-term presence of VCR or DOX not only presented changes in the resistance phenotype but also variations in their growth and metastatic pattern.

Cancer cells may frequently develop cross-resistance to structurally dissimilar chemotherapeutic agents. However, the molecular mechanisms for sensitivity and resistance of tumor cells towards chemotherapy are still partially understood. Antineoplasic drugs have been shown to induce apoptosis in chemosensitive leukemias and solid tumors. In this work, cross-resistance among vincristine (VCR), doxorubicin (DOX) and other antineoplasic agents commonly used in the treatment of leukemia such as etoposide (VP-16), methotrexate (MTX), cyclophosphamide (CTX), dexamethasone (DEX), cytarabine (Ara-C) and L-asparaginase on vincristine resistant (LBR-V160), doxorubicin resistant (LBR-D160) and sensitive (LBR-) murine leukemic T cell lines, was determined. The effect of antineoplasic agents was assayed by tritiated thymidine incorporation. Our results showed that VCR exhibited cross-resistance with DOX, VP-16, DEX and MTX, while DOX demonstrated cross-resistance with VCR, VP-16 and MTX. Ara-C failed to present cross-resistance with any cell line. Apoptosis induced by the above drugs on the same cell lines was analyzed by acridine orange and ethidium bromide staining, DNA hypoploidy (flow cytometry) and oligonucleosomal fragmentation of nuclear DNA showing that therapeutic concentrations of these chemotherapeutic agents induced apoptosis in the LBR- cell line. Our results demonstrated that, except for DEX, none of the drugs presenting cross-resistance were able to induce cell death on LBR-V 160 or LBR-D 160 cell lines.

CD44 is a widely distributed set of cell surface glycoproteins expressed in several types of cells and tissues, implicated in cell-cell and cell-substrate interactions. This molecule plays a major role in cell differentiation, development and activation and has also been described as a potential marker of malignancy and metastasis. In the present study we investigated by RT-PCR followed by exon specific amplification the expression of CD44 splice variants in four different murine tumors as well as in the invaded organs in order to correlate the expression of CD44 variants with potential tumor invasiveness and their implications for growth. Our data showed deregulation in the expression of CD44 isoforms but no discernible correlation in isoform expression pattern. However, in all tumors studied isoforms presented by the primary tumor were detected in the invaded organs before metastasis could be demonstrated by histopathological analysis.

CD44 is an adhesion molecule involved in many biological functions and has been described to play a role in tumor progression as well as in promotion of metastasis. It has also been suggested that expression of certain CD44 isoforms could be useful for breast and ovarian cancer screening, detection or staging. However, many other reports document no correlation between CD44 isoform expression and tumor malignancy. In light of such contradictory findings, we evaluated by exon-specific RT-PCR whether the expression of CD44 isoforms in breast and ovarian tumors correlated with any of the diagnostic criteria used to assess these diseases. We found a deregulation in the CD44 expression pattern in malignant tumors of both type of cancer compared with the one in benign tumors or normal tissue. However, we could not find a clear correlation between this deregulation or a given CD44 isoform and any diagnostic criteria evaluated, such as age, clinical data, tumor size, hormone receptor status, histological grade or aggressiveness.

Multidrug resistance (MDR) lines from a murine T-cell leukemia were selected in increasing vincristine (VCR) or doxorubicin (DOX) concentrations. Daunorubicin (DNR) efflux was evidenced after 25 additional passages with constant 160 ng ml(-1) of either VCR or DOX, an effect that was inhibited by verapamil, cyclosporin-A (CsA) and PSC 833. The expression of Pgp was not evidenced in the resistant cell lines using anti-human Pgp antibodies. Cell proliferation assay showed that cell lines resistant to VCR (LBR-V160) or DOX (LBR-D160) required higher doses of either drug to produce GI50 compared with control cell line obtained after culture in the absence of VCR or DOX. When resistant cell lines were maintained during 60 days in the absence of either VCR or DOX, MDR phenotype reversal was obtained in LBR-D160 while LBR-V160 remained resistant to the drug, as shown by cell proliferation assays and by drug efflux pump functionality. When VCR or DOX were used together with either CsA or PSC 833, the latter was more effective to produce reversal of resistance than the former, whereas CsA presented greater cytotoxic effect than PSC 833 for sensitive and resistant cells. Cross-resistance was found between VCR, DOX and other antineoplasic agents on murine leukemic cell line. VCR was more effective to induce MDR since the resistant cell lines were more stable to the MDR phenotype.

Bothrops ammodytoides, the smallest representative of this genus, is found only in Argentina. Venom was extracted from thirty adult specimens (35-70 cm in length, 90-300 g in weight) captured in the Province of Buenos Aires and kept in captivity. Venom yield was 3-30 mg. SDS-PAGE showed strong bands at 14.0; 23-25; 45; 54 and 63 kDa and weak bands at 17.0; 30.0; 40.0 and 85.0 kDa. Toxic activities were: LD50 (intravenous, mice) 0.5+/-0.2 microg/g; minimal procoagulant dose on human plasma (MPD-P) 35+/-2 mg/l; and minimal defibrinogenating dose (MDD, mice) 6-12 microg. Hemorrhagic and/or necrotic activities appear to play a major role in lethality; minimal hemorrhagic dose (MHD, mice) is 10+/-2 microg/g and minimal necrotizing dose (MND, mice) is 38+/-5 microg. The LD50, MPD-P and MND are among the lowest in venoms from Bothrops species found in Argentina. B. ammodytoides venom exhibited high proteolytic and phospholipase A2 activities. Most of the B. ammodytoides venom components cross-react with Bivalent Bothropic antivenom (Instituto Nacional de Producción de Biológicos ANLIS Dr. G. Malbrin, against B. alternatus and B. neuwiedii venoms). One ml of antivenom neutralizes 1.2 mg of B. ammodytoides venom.

We have established and characterized a cell line (LBL) from a spontaneous murine T lymphoma LB. Histopathological analysis has demonstrated LB primary tumor infiltration in spleen, lymph nodes, liver, thymus, bone marrow and lung. However LBL cells infiltrated all these organs except lung. Two sublines with different growth behavior were derived from LBL cell line. One of them grew in suspension as clusters (LBLc) while the other one grew as adherent monolayers (LBLa). Growth rate, response to mitogenic stimuli and apoptosis induction were different among the parental cell line and the derived sublines. CD44 was expressed constitutively in LBL and LBLa cells. In contrast LBLc cells only expressed similar levels of this molecule when stimulated with PMA. LBLa cells showed hyaluronic acid (HA) binding properties, while LBL and LBLc cells were not able to bind HA even when activated with PMA. We postulate that differences in HA binding could be related with different infiltration behaviors.

Gut ischemia-reperfusion (G-IR) induces a systemic inflammatory response, in which leukocyte contribution to this injury in distant organs is important. ICAM-1 as well as CD11/CD18 have been involved in leukocyte infiltration in liver and lungs. CD44 adhesion molecule plays an essential role in other inflammatory processes such as rheumatoid arthritis and allergic contact dermatitis, however its implication in G-IR has not been described. In order to establish a possible role of CD44 in the development of systemic inflammation by G-IR, we have studied CD44 mRNA expression by RT-PCR in a murine model of gut ischemia reperfusion. Animals subjected to G-IR showed an increased number of CD44 variable isoforms expressed in liver and spleen compared to non-treated animals or animals subjected to laparotomy. This finding indicates that G-IR specifically induces the expression of different CD44 variable isoforms. Liver CD44 upregulation in animals subjected to G-IR suggests a contribution of this molecule to lymphocyte activation and migration to this injured organ. Moreover, increased isoform expression in spleen may be induced by the proinflammatory environment resulting from a systemic depuration activity.

We have studied the immunochemical cross-reactivity and cross-neutralization of the lethal potency, hemorrhagic, necrotizing, procoagulant and (indirect) hemolytic activities of Bothrops jararacussu venom by the standard antivenoms produced in Argentina. These antivenoms are horse immunoglobulin F (ab')2 fragments from animals immunized with 1) Crotalus durissus terrificus venom (Monovalent Anticrotalic antivenom); 2) Bothrops alternatus and B. neuwiedii venoms (Bivalent Botropic antivenom); 3) B. alternatus, B. neuwiedii, B. jararaca and B. jararacussu venoms (Tetravalent Bothropic, or "Misiones" antivenom) and 4) B. alternatus, B. neuwiedii and C. d. terrificus venoms (Trivalent Botropic-Crotalic antivenom). In preincubation experiments, all the heterologous antivenoms neutralized the toxic and biological activities of B. jararacussu venom with a potency at least as high as the Tetravalent Botropic (i.e. the only homologous) antivenom, in which B. jararacussu venom was included as immunogen. These results suggest the possibility of using heterologous antibothropic antivenoms for the treatment of snake bites by B. jararacussu.

CD44 is a widely expressed cell-surface transmembrane glycoprotein involved in diverse adhesive processes. Its isoforms have been implicated in tumor progression and are considered a promising marker for evaluation of the metastatic potential of various tumors. Several methods have been described for the analysis of CD44 isoforms in tumor cells, including immuno-histochemistry, RT-PCR followed by hybridization and nested RT-PCR. We describe an alternative nested PCR for the analysis of CD44 isoform expression in various malignancies. Total RNA was isolated from various shock-frozen tissues from human tumors, reverse-transcribed and PCR-amplified using CD44-specific primers. Reverse-transcription was performed by two different methods, either using Tth-polymerase or MMLV-RT. Exon-specific amplification was then carried out using specific primers for each variable exon. Amplification products were assayed by agarose gel electrophoresis. Comparison of the patterns obtained from the first amplification and from the exon-specific amplification allowed to identify exons expressed by tumor tissues, as well as the genomic organization of CD44 isoforms. The method developed proved to be sensitive, reliable and inexpensive in comparison with other methods. It can be performed even in solid tumors and for numerous samples, and is suitable for laboratories with limited resources.

Ligaria cuneifolia (R. et P.) Tiegh. is an hemiparasite species used in Argentine folk medicine as a substitute for the European mistletoe (Viscum album L.) based on its putative activity of decreasing high blood pressure. This paper analyzes flavonoid composition, protein constituents and the possible immunomodulatory and antitumoral effects of this species. Micromolecular study disclosed quercetin-free, quercetin-glycosylated and proanthocyanidins corresponding to cyanidin monomers, which implies a particular metabolic pathway. Proteins present in L. cuneifolia extracts analyzed by SDS-PAGE presented multiple bands with molecular weights ranging from 14 to 90 kD. These features contribute to the characterization of the native mistletoe. As V. album is being used in cancer treatment due to its immunomodulatory and antitumoral activity, the action of aqueous L. cuneifolia extracts on murine lymphocytes was investigated. Culture of murine spleen cells alone or stimulated with Concanavalin A or lipopolysaccharide in presence of L. cuneifolia extracts indicated a certain stimulation of splenocytes alone and an inhibition of splenocytes stimulated with Concanvalin A or lipopolysaccharide. An inhibitory effect was also observed on the proliferation of murine leukemia cells. In addition, aqueous extracts increased nitric oxide production by murine macrophages. These results suggest that L. cuneifolia extracts exert an immunomodulatory effect on the mouse immune system.

The immunochemical cross-reactivity and neutralizing capacity of four crotalinae antivenoms consisting in equine F(ab')2 fragments and available in Argentina (bothropic Bivalent, against Bothrops alternatus and B. neuwiedii venoms; bothropic Tetravalent, against B. alternatus, B. neuwiedii; B. jararaca and B. jararacussu venoms; bothropic crotalic Trivalent, against B. alternatus, B. neuwiedii and Crotalus (C.) durissus terrificus venoms and anticrotalic against C. d. terrificus venom) were studied against B. alternatus, B. ammodytoides; B. jararaca; B. jararacussu, B. moojeni; B. neuwiedii and C. d. terrificus venoms. SDS-PAGE analysis of the Bothrops venoms showed protein bands of high (>40 kDa) medium (20-40 kDa) and low (<15 kDa) molecular weights, while that of C. d. terrificus exhibited a large amount of material with molecular weight of 15.0 kDa or lower. Immunoblotting showed a high cross-reactivity of all the major protein bands with all the antivenoms (even heterologous) tested. All the antivenoms were effective in neutralizing the lethal activity of the venoms tested, and in some cases (B. jararaca and B. jararacussu) heterologous antivenoms exhibited similar neutralizing capacity than the homologous ones. In spite of the differences in biochemical composition and pharmacology, Bothropic antivenoms displayed a significant neutralizing capacity on lethal activity of C. d. terrificus venom. In addition, all the antivenoms (including the anticrotalic) were highly effective in neutralizing the hemorragic, necrotizing, procoagulant, and proteolytic activities. The antivenoms tested produced different degrees of inhibition of phospholipase A2 activity, which exhibited a certain specificity but was also related to the enzyme content in the venom.

In the production of current therapeutic antisera used in Argentina for Bothrops snakebite, the Bothrops moojeni venom is not used as immunogen, since this snake is not considered a serious public health problem. Accidents caused by this species have not been reported in this country even though Bothrops moojeni is not unfrequent in some regions of Misiones. Despite the high degree of immunological cross reactivity found among the Crotalinae venoms and, in this particular case, among the venoms from the Bothrops Genus, there exists a significant intraspecific variation in venom composition, particularly in specimens arising from different geographic regions. In this study, the antivenoms prepared at the Instituto Nacional de Producción de Biológicos A.N.L.I.S. Dr. Carlos G. Malbrán have been tested for their immunochemical cross reactivity and neutralizing ability of enzymatic and toxic activities of venom from Argentinian specimens of Bothrops moojeni from Misiones. Immunological cross-reactivity was tested by double-immunoprecipitation, immunoelectrophoresis, Western-blot and ELISA. Neutralizing ability of antivenoms against proteolytic, indirect hemolytic activity, procoagulant activity, he-morrhagic activity and necrotizing activity. The Lethal Dose 50 was 1.5 mg/kg body weight; this value is located in the range to those obtained with the venom from Brazilian specimens. It was observed that all the antivenoms exhibited a strong immunochemical cross reactivity and that they were able to neutralize in different degree both, enzymatic and toxic activities of B. moojeni venom. From these results, it can be assumed that the antivenom tested could be employed successfully in cases of B. moojeni snakebites.

LB leukemia is a nonimmunogenic T cell tumor which spontaneously arose in a BALB/c mouse; efforts to induce immunological rejection of the leukemic cells have always failed. The leukemic cells grow rapidly and progressively in the syngeneic host invading spleen, lymph nodes and liver. A cell line (LBC) was developed from the original tumor. Both the original tumor and the cell line have been characterized as expressing the Thy 1+, CD3-, CD25+, MHC class I+, class II-, CD4- (original tumor), CD4+ (cell line), CD8+, gp70-, J11d.2+ phenotypes. Immunization of syngeneic mice with irradiated LBC cells induced cytotoxic T lymphocytes as well as anti-LBC antibodies which reacted with components of 14, 16 and 27 kDa present on LB tumor cells, LBC cell line and normal thymocytes but not on normal lymph node cells. Immunization of syngeneic mice with LBC cells partially protected them against subsequent challenge with the original tumor cells. The effect of sera from tumor-bearing mice and the super-natants from short term cultures were studied on cell proliferation. An inhibitory activity was demonstrated in these fluids, which was abrogated by addition of exogenous IL-2. ELISA showed the presence of soluble IL-2R alpha chain both in the conditioned medium as well as in the serum, which was demonstrated to be responsible for the inhibitory activity. The soluble IL-2R was produced by LB leukemic cells and exerted the inhibitory activity blocking cell proliferation and modulating immune response by binding to free IL-2. Using reverse-transcription PCR, mRNA for IL-2 was found to be present in tumor cells. Our findings indicate that LB cell proliferation is mediated by an autocrine pathway involving endogenous IL-2 generation, despite the fact that these cells are not dependent on exogenous IL-2 to grow in culture. The relationship between tumorigenicity and expression of MHC class II was also investigated. In vitro treatment with IFN-gamma failed to induce the expression of class II antigens in LBC cell line. Therefore these cells were tri-transfected by a liposome-mediated protocol with 1-A alpha d, I-A beta d genes and pSV2neo. Cells were selected to grow in medium containing Genetecin (G418) and surviving transfectants were cloned. Three I-A+ clones were obtained (LBCT) and were used to induce a specific CTL response against tumor cells. Syngeneic mice inoculated with 10(3) LBCT cells failed to develop a tumor while the DT50 of mice injected with 10(6) LBCT cells was three times the value for mice injected with LBC cells (I-A-). It is suggested that neoexpression of MHC class II molecules enhances anti-tumor response by transforming tumor cells into professional antigen-presenting cells, which may be used to improve tumor-specific immunity in the autologous host.

Induction of anti-tumour immunity in syngeneic mice by LBC cell line derived from a non-immunogenic T cell leukaemia was studied. The immunization of BALB/c mice with LBC irradiated cells induced in them anti-tumour spleen cells, cytotoxic T lymphocytes and anti-LBC antibodies. The anti-LBC antibodies reacted with components of 14, 16 and 27 kDa present on LB tumour cells, LBC cell line and normal thymocytes, but not with normal lymph node cells. Furthermore, immunization of the autologous hosts with LBC cells partially protected them against subsequent challenge with the original LB leukaemic cells. These findings demonstrate that culture conditions induced modifications in the antigenic properties of the leukaemic cells, allowing LBC cells to stimulate an immune response directed against components expressed at early stages during T cell maturation. These results also suggest that the immune response is responsible for the prolongation of the survival time of the mice inoculated with the parental leukaemic cells.

The effect of sera from mice bearing a T cell lymphoid leukaemia (LB) and the supernatants from short term cultures of the tumour cells were studied on cell proliferation using syngeneic and allogeneic normal and tumour cells. An inhibitory activity was demonstrated in 24-48 h supernatants of LB cells in culture and disappeared after 4 days of culture. Inhibitory activity was cytostatic but not cytotoxic and was non-specific since it inhibited the growth of both syngeneic and allogeneic normal and tumour cells. Such activity was found in the 10(5)-1.3 x 10(5) M(r) serum fraction after a Sephacryl S200 chromatography. Though sensitive to protease, trypsin or neuraminidase treatment, which indicated its glycoprotein nature, it remained stable after heating or freezing-thawing cycles as well as after alkaline, acid or hyaluronidase treatment. Addition of exogenous IL-2 abrogated inhibitory activity. ELISA showed the presence of soluble IL-2R both in LB conditioned medium and in above serum fraction. It is demonstrated that the inhibitory factor, soluble IL-2R, is produced by LB leukaemia cells, then secreted into blood and ascitic fluid or released into culture supernatants. Soluble IL-2R exerts inhibitory activity blocking cell proliferation and modulating immune response by binding to free IL-2.