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Diffuse gliomas are tumors that arise from glial or glial progenitor cells that have historically been classified as low-grade glioma (LGG), a slower-growing tumor, and glioblastoma (GBM), a more aggressive tumor. Despite advances in the diagnosis and treatment of glioma, the median survival time after diagnosis of GBM remains low, approximately 15 months, and with a 5-year overall survival rate of only 6.8%. Therefore, new biomarkers that could support earlier diagnosis and prognosis of these tumors would be of great value. MUC17, a member of the membrane-bound mucins, has been identified as a potential biomarker in several tumors. However, this mucin is unexplored in adult gliomas. Here we have shown for the first time in a retrospective study and byin silicoanalysis that MUC17 is one of the relevant mutant genes in adult gliomas and that the increase in MUC17 methylation correlates with the increase in glioma grade malignancy. Patients with MUC17 mutation had a poorer prognosis compared to their wild-type counterparts in both the LGG and GBM cohorts. We also analyzed which mutational profile correlates more strongly with poor survival. Therefore, in the present work, we present a new potential biomarker to be investigated for the diagnosis and prognosis in adult diffuse glioma.Key pointsMUC17 accounts the mutational burden in gliomasMutated MUC17 is associated with poor prognosis in both LGG and GBM cohortsMUC17 methylation rate increases with tumor grade in gliomasThe C > T base change is the most common missense MUC17 mutation in GBM and correlates with a poorer prognosis.

Glioblastoma (GBM) is the most common adult primary tumor of the CNS characterized by rapid growth and diffuse invasiveness into the brain parenchyma. The GBM resistance to chemotherapeutic drugs may be due to the presence of cancer stem cells (CSCs). The CSCs activate the same molecular pathways as healthy stem cells such as WNT, Sonic hedgehog (SHH), and Notch. Mutations or deregulations of those pathways play a key role in the proliferation and differentiation of their surrounding environment, leading to tumorigenesis. Here we investigated the effect of SHH signaling pathway inhibition in human GBM cells by using GANT-61, considering stem cell phenotype, cell proliferation, and cell death. Our results demonstrated that GANT-61 induces apoptosis and autophagy in GBM cells, by increasing the expression of LC3 II and cleaved caspase 3 and 9. Moreover, we observed that SHH signaling plays a crucial role in CSC phenotype maintenance, being also involved in the epithelial-mesenchymal transition (EMT) phenotype. We also noted that SHH pathway modulation can regulate cell proliferation as revealed through the analysis of Ki-67 and c-MYC expressions. We concluded that SHH signaling pathway inhibition may be a promising therapeutic approach to treat patients suffering from GBM refractory to traditional treatments.

Background/aim Glioblastoma (GB) is the most aggressive type of tumor in the central nervous system and is characterized by resistance to therapy and abundant vasculature. Tumor vessels contribute to the growth of GB, and the tumor microenvironment is thought to influence tumor vessels. We evaluated the molecular communication between human GB cells and human brain microvascular endothelial cells (HBMEC) in vitro. Materials and methods We investigated whether GB-conditioned media (GB-CM) influenced HBMEC proliferation and migration, as well as the levels of MMP-9, CXCL12, CXCR4, CXCR7, VEGFs, VEGFR-2, and WNT5a in HBMEC. Results Although HBMEC proliferation was not modified, increased HBMEC migration was detected after GB-CM treatment. Furthermore, treatment of HBMEC with GB-CM resulted in increased levels of MMP-9 and CXCR4. The levels of WNT5a, VEGFs and VEGFR-2 were not affected. Conclusion GB-secreted factors lead to increased endothelial cell migration and to increased levels of MMP-9 and CXCR4.

MUC16 is a member of the attached mucin family that encodes cancer antigen 125 (CA-125), but the association of MUC16 status with grade and subtypes of glioma patients has not yet been established. Data for MUC16 mRNA expression in 37 different cancer types were considered, and genomic data from the Cancer Genome Atlas (TCGA) from 1051 low-grade glioma (LGG) patients and 833 glioblastoma (GBM) patients were analyzed. LGG and GBM has low expression of MUC16, but it is frequently mutated in GBM. Kaplan-Meier survival analysis, glioma subtypes, methylation, and isocitrate dehydrogenase (IDH1) status were all performed. We found that mutated-MUC16 in LGG patients is associated with better prognosis considering overall survival (OS), IDH1, methylation, 1p/19q, and 10q status. Conversely, MUC16 mutation were related with worse prognosis in GBM patients upon analyzing those same parameters. Therefore, MUC16 mutations may assist in glioma diagnosis and prognosis and should be further studied in this tumor type.

Circulating tumor DNA (ctDNA) in fluids has gained attention because ctDNA seems to identify tumor-specific abnormalities, which could be used for diagnosis, follow-up of treatment, and prognosis: the so-called liquid biopsy. Liquid biopsy is a minimally invasive approach and presents the sum of ctDNA from primary and secondary tumor sites. It has been possible not only to quantify the amount of ctDNA but also to identify (epi)genetic changes. Specific mutations in genes have been identified in the plasma of patients with several types of cancer, which highlights ctDNA as a possible cancer biomarker. However, achieving detectable concentrations of ctDNA in body fluids is not an easy task. ctDNA fragments present a short half-life, and there are no cut-off values to discriminate high and low ctDNA concentrations. Here, we discuss the use of ctDNA as a cancer biomarker, the main methodologies, the inherent difficulties, and the clinical predictive value of ctDNA.

Glioblastoma (GBM) is the most common adult primary tumor of the CNS characterized by rapid growth and diffuse invasiveness into the brain parenchyma. The GBM resistance to chemotherapeutic drugs may be due to the presence of cancer stem cells (CSCs). The CSCs activate the same molecular pathways as healthy stem cells such as WNT, Sonic hedgehog (SHH), and Notch. Mutations or deregulations of those pathways play a key role in the proliferation and differentiation of their surrounding environment, leading to tumorigenesis. Here we investigated the effect of SHH signaling pathway inhibition in human GBM cells by using GANT-61, considering stem cell phenotype, cell proliferation, and cell death. Our results demonstrated that GANT-61 induces apoptosis and autophagy in GBM cells, by increasing the expression of LC3 II and cleaved caspase 3 and 9. Moreover, we observed that SHH signaling plays a crucial role in CSC phenotype maintenance, being also involved in the epithelial-mesenchymal transition (EMT) phenotype. We also noted that SHH pathway modulation can regulate cell proliferation as revealed through the analysis of Ki-67 and c-MYC expressions. We concluded that SHH signaling pathway inhibition may be a promising therapeutic approach to treat patients suffering from GBM refractory to traditional treatments.

Glioblastoma is an extremely aggressive and deadly brain tumor known for its striking cellular heterogeneity and capability to communicate with microenvironment components, such as microglia. Microglia-glioblastoma interaction contributes to an increase in tumor invasiveness, and Wnt signaling pathway is one of the main cascades related to tumor progression through changes in cell migration and invasion. However, very little is known about the role of canonical Wnt signaling during microglia-glioblastoma crosstalk. Here, we show for the first time that Wnt3a is one of the factors that regulate interactions between microglia and glioblastoma cells. Wnt3a activates the Wnt/β-catenin signaling of both glioblastoma and microglial cells. Glioblastoma-conditioned medium not only induces nuclear translocation of microglial β-catenin but also increases microglia viability and proliferation as well as Wnt3a, cyclin-D1, and c-myc expression. Moreover, glioblastoma-derived Wnt3a increases microglial ARG-1 and STI1 expression, followed by an upregulation of IL-10 mRNA levels, and a decrease in IL1β gene expression. The presence of Wnt3a in microglia-glioblastoma co-cultures increases the formation of membrane nanotubes accompanied by changes in migration capability. In vivo, tumors formed from Wnt3a-stimulated glioblastoma cells presented greater microglial infiltration and more aggressive characteristics such as growth rate than untreated tumors. Thus, we propose that Wnt3a belongs to the arsenal of factors capable of stimulating the induction of M2-like phenotype on microglial cells, which contributes to the poor prognostic of glioblastoma, reinforcing that Wnt/β-catenin pathway can be a potential therapeutic target to attenuate glioblastoma progression.

An outstanding characteristic of gliomas is their infiltration into brain parenchyma, a property that impairs complete surgical resection; consequently, these tumors might recur, resulting in a high mortality rate. Gliomas invade along preferential routes, such as those along white matter tracts and in the perineuronal and perivascular spaces. Brain extracellular components and their partners and modulators play a crucial role in glioma cell invasion. This review presents an extensive survey of the literature, showing how the brain extracellular matrix (ECM) is modulated during the glioma infiltration process. We explore aspects of ECM interaction with glioma cells, reviewing the main glycosaminoglycans, glycoproteins and proteoglycans. We discuss the roles of ECM-binding proteins, including CD44, RHAMM, integrins and axonal guidance molecules, and highlight the role of proteases and glycosidases in glioma infiltration; in binding and release chemokines, cytokines and growth factors; and in generating new bioactive ECM fragments. We also consider the roles of cytoskeletal signaling, angiogenesis, miRNAs and the glial-to-mesenchymal transition linked to glioma invasion. We closely discuss therapeutic approaches based on the modulation of the extracellular matrix, targeting the control of glioma infiltration, its relative failure in clinical trials, and potential means to overcome this difficulty.

Glioblastoma (GBM) is the most malignant primary brain tumor, with an average survival rate of 15 months. GBM is highly refractory to therapy, and such unresponsiveness is due, primarily, but not exclusively, to the glioma stem-like cells (GSCs). This subpopulation express stem-like cell markers and is responsible for the heterogeneity of GBM, generating multiple differentiated cell phenotypes. However, how GBMs maintain the balance between stem and non-stem populations is still poorly understood. We investigated the GBM ability to interconvert between stem and non-stem states through the evaluation of the expression of specific stem cell markers as well as cell communication proteins. We evaluated the molecular and phenotypic characteristics of GSCs derived from differentiated GBM cell lines by comparing their stem-like cell properties and expression of connexins. We showed that non-GSCs as well as GSCs can undergo successive cycles of gain and loss of stem properties, demonstrating a bidirectional cellular plasticity model that is accompanied by changes on connexins expression. Our findings indicate that the interconversion between non-GSCs and GSCs can be modulated by extracellular factors culminating on differential expression of stem-like cell markers and cell-cell communication proteins. Ultimately, we observed that stem markers are mostly expressed on GBMs rather than on low-grade astrocytomas, suggesting that the presence of GSCs is a feature of high-grade gliomas. Together, our data demonstrate the utmost importance of the understanding of stem cell plasticity properties in a way to a step closer to new strategic approaches to potentially eliminate GSCs and, hopefully, prevent tumor recurrence.

Glioblastomas (GBM) comprise 17% of all primary brain tumors. These tumors are extremely aggressive due to their infiltrative capacity and chemoresistance, with glial-to-mesenchymal transition (GMT) proteins playing a prominent role in tumor invasion. One compound that has recently been used to reduce the expression of these proteins is shikonin (SHK), a naphthoquinone with anti-tumor properties. Temozolomide (TMZ), the most commonly used chemotherapeutic agent in GBM treatment, has so far not been studied in combination with SHK. Here, we investigated the combined effects of these two drugs on the proliferation and motility of GBM-derived cells. The cytotoxic and proliferative effects of SHK and TMZ on human GBM-derived cells were tested using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Ki67 staining and BrdU incorporation assays. The migration capacities of these cells were evaluated using a scratch wound assay. The expression levels of β3 integrin, metalloproteinases (MMPs) and GMT-associated proteins were determined by Western blotting and immunocytochemistry. We found that GBM-derived cells treated with a combination of SHK and TMZ showed decreases in their proliferation and migration capacities. These decreases were followed by the suppression of GMT through a reduction of β3 integrin, MMP-2, MMP-9, Slug and vimentin expression via inactivation of PI3K/AKT signaling. From our results we conclude that dual treatment with SHK and TMZ may constitute a powerful new tool for GBM treatment by reducing therapy resistance and tumor recurrence.

Brown spider (Loxosceles genus) bites have been reported worldwide. The venom contains a complex composition of several toxins, including phospholipases-D. Native or recombinant phospholipase-D toxins induce cutaneous and systemic loxoscelism, particularly necrotic lesions, inflammatory response, renal failure, and hematological disturbances. Herein, we describe the cloning, heterologous expression and purification of a novel phospholipase-D toxin, LiRecDT7 in reference to six other previously described in phospholipase-D toxin family. The complete cDNA sequence of this novel brown spider phospholipase-D isoform was obtained and the calculated molecular mass of the predicted mature protein is 34.4 kDa. Similarity analyses revealed that LiRecDT7 is homologous to the other dermonecrotic toxin family members particularly to LiRecDT6, sharing 71% sequence identity. LiRecDT7 possesses the conserved amino acid residues involved in catalysis except for a conservative mutation (D233E) in the catalytic site. Purified LiRecDT7 was detected as a soluble 36 kDa protein using anti-whole venom and anti-LiRecDT1 sera, indicating immunological cross-reactivity and evidencing sequence-epitopes identities similar to those of other phospholipase-D family members. Also, LiRecDT7 exhibits sphingomyelinase activity in a concentration dependent-manner and induces experimental skin lesions with swelling, erythema and dermonecrosis. In addition, LiRecDT7 induced a massive inflammatory response in rabbit skin dermis, which is a hallmark of brown spider venom phospholipase-D toxins. Moreover, LiRecDT7 induced in vitro hemolysis in human erythrocytes and increased blood vessel permeability. These features suggest that this novel member of the brown spider venom phospholipase-D family, which naturally contains a mutation (D233E) in the catalytic site, could be useful for future structural and functional studies concerning loxoscelism and lipid biochemistry. Highlights: 1- Novel brown spider phospholipase-D recombinant toxin contains a conservative mutation (D233E) on the catalytic site. 2-LiRecDT7 shares high identity level with isoforms of Loxosceles genus. 3-LiRecDT7 is a recombinant protein immunodetected by specific antibodies to native and recombinant phospholipase-D toxins. 4-LiRecDT7 shows sphingomyelinase-D activity in a concentration-dependent manner, but less intense than other isoforms. 5-LiRecDT7 induces dermonecrosis and inflammatory response in rabbit skin. 6-LiRecDT7 increases vascular permeability in mice. 7-LiRecDT7 triggers direct complement-independent hemolysis in erythrocytes. J. Cell. Biochem. 114: 2479–2492, 2013. © 2013 Wiley Periodicals, Inc.

Loxosceles spiders are responsible for serious human envenomations worldwide. The collection of symptoms found in victims after accidents is called loxoscelism and is characterized by two clinical conditions: cutaneous loxoscelism and systemic loxocelism. The only specific treatment is serum therapy, in which an antiserum produced with Loxosceles venom is administered to the victims after spider accidents. Our aim was to improve our knowledge, regarding the immunological relationship among toxins from the most epidemiologic important species in Brazil (Loxosceles intermedia, Loxosceles gaucho and Loxosceles laeta). Immunoassays using spider venoms and L. intermedia recombinant toxins were performed and their cross-reactivity assessed. The biological conservation of the main Loxosceles toxins (Phospholipases-D, Astacin-like metalloproteases, Hyaluronidase, ICK-insecticide peptide and TCTP-histamine releasing factor) were investigated. An in silico analysis of the putative epitopes was performed and is discussed on the basis of the experimental results. Our data is an immunological investigation in light of biological conservation throughout the Loxosceles genus. The results bring out new insights on brown spider venom toxins for study, diagnosis and treatment of loxoscelism and putative biotechnological applications concerning immune conserved features in the toxins.

The Loxosceles genus spiders (the brown spiders) are encountered in all the continents, and the clinical manifestations following spider bites include skin necrosis with gravitational lesion spreading and occasional systemic manifestations, such as intravascular hemolysis, thrombocytopenia and acute renal failure. Brown spider venoms are complex mixtures of toxins especially enriched in three molecular families: the phospholipases D, astacin-like metalloproteases and Inhibitor Cystine Knot (ICK) peptides. Other toxins with low level of expression also present in the venom include the serine proteases, serine protease inhibitors, hyaluronidases, allergen factors and translationally controlled tumor protein (TCTP). The mechanisms by which the Loxosceles venoms act and exert their noxious effects are not fully understood. Except for the brown spider venom phospholipase D, which causes dermonecrosis, hemolysis, thrombocytopenia and renal failure, the pathological activities of the other venom toxins remain unclear. The objective of the present review is to provide insights into the brown spider venoms and loxoscelism based on recent results. These insights include the biology of brown spiders, the clinical features of loxoscelism and the diagnosis and therapy of brown spider bites. Regarding the brown spider venom, this review includes a description of the novel toxins revealed by molecular biology and proteomics techniques, the data regarding three-dimensional toxin structures, and the mechanism of action of these molecules. Finally, the biotechnological applications of the venom components, especially for those toxins reported as recombinant molecules, and the challenges for future study are discussed.

Brown spider (Loxosceles genus) bites have been reported worldwide. The venom contains a complex composition of several toxins, including phospholipases-D. Native or recombinant phospholipase-D toxins induce cutaneous and systemic loxoscelism, particularly necrotic lesions, inflammatory response, renal failure, and hematological disturbances. Herein, we describe the cloning, heterologous expression and purification of a novel phospholipase-D toxin, LiRecDT7 in reference to six other previously described in phospholipase-D toxin family. The complete cDNA sequence of this novel brown spider phospholipase-D isoform was obtained and the calculated molecular mass of the predicted mature protein is 34.4 kDa. Similarity analyses revealed that LiRecDT7 is homologous to the other dermonecrotic toxin family members particularly to LiRecDT6, sharing 71% sequence identity. LiRecDT7 possesses the conserved amino acid residues involved in catalysis except for a conservative mutation (D233E) in the catalytic site. Purified LiRecDT7 was detected as a soluble 36 kDa protein using anti-whole venom and anti-LiRecDT1 sera, indicating immunological cross-reactivity and evidencing sequence-epitopes identities similar to those of other phospholipase-D family members. Also, LiRecDT7 exhibits sphingomyelinase activity in a concentration dependent-manner and induces experimental skin lesions with swelling, erythema and dermonecrosis. In addition, LiRecDT7 induced a massive inflammatory response in rabbit skin dermis, which is a hallmark of brown spider venom phospholipase-D toxins. Moreover, LiRecDT7 induced in vitro hemolysis in human erythrocytes and increased blood vessel permeability. These features suggest that this novel member of the brown spider venom phospholipase-D family, which naturally contains a mutation (D233E) in the catalytic site, could be useful for future structural and functional studies concerning loxoscelism and lipid biochemistry.1- Novel brown spider phospholipase-D recombinant toxin contains a conservative mutation (D233E) on the catalytic site. 2-LiRecDT7 shares high identity level with isoforms of Loxosceles genus. 3-LiRecDT7 is a recombinant protein immunodetected by specific antibodies to native and recombinant phospholipase-D toxins. 4-LiRecDT7 shows sphingomyelinase-D activity in a concentration-dependent manner, but less intense than other isoforms. 5-LiRecDT7 induces dermonecrosis and inflammatory response in rabbit skin. 6-LiRecDT7 increases vascular permeability in mice. 7-LiRecDT7 triggers direct complement-independent hemolysis in erythrocytes.

Secretaria de Estado de Ciencia Tecnologia e Ensino Superior (SETI) do Parana Fundacao Araucaria-PR Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Brown spider dermonecrotic toxins (phospholipases-D) are the most well-characterized biochemical constituents of Loxosceles spp. venom. Recombinant forms are capable of reproducing most cutaneous and systemic manifestations such as dermonecrotic lesions, hematological disorders, and renal failure. There is currently no direct confirmation for a relationship between dermonecrosis and inflammation induced by dermonecrotic toxins and their enzymatic activity. We modified a toxin isoform by site-directed mutagenesis to determine if phospholipase-D activity is directly related to these biological effects. the mutated toxin contains an alanine substitution for a histidine residue at position 12 (in the conserved catalytic domain of Loxosceles intermedia Recombinant Dermonecrotic Toxin - LiRecDT1). LiRecDT1H12A sphingomyelinase activity was drastically reduced, despite the fact that circular dichroism analysis demonstrated similar spectra for both toxin isoforms, confirming that the mutation did not change general secondary structures of the molecule or its stability. Antisera against whole venom and LiRecDT1 showed cross-reactivity to both recombinant toxins by ELISA and immunoblotting. Dermonecrosis was abolished by the mutation, and rabbit skin revealed a decreased inflammatory response to LiRecDT1H12A compared to LiRecDT1. Residual phospholipase activity was observed with increasing concentrations of LiRecDT1H12A by dermonecrosis and fluorometric measurement in vitro. Lipid arrays showed that the mutated toxin has an affinity for the same lipids LiRecDT1, and both toxins were detected on RAEC cell surfaces. Data from in vitro choline release and HPTLC analyses of LiRecDT1-treated purified phospholipids and RAEC membrane detergent-extracts corroborate with the morphological changes. These data suggest a phospholipase-D dependent mechanism of toxicity, which has no substrate specificity and thus utilizes a broad range of bioactive lipids. (C) 2010 Elsevier B.V. All rights reserved. Univ Fed Parana, Dept Cell Biol, BR-81531990 Curitiba, Parana, Brazil Universidade Federal de São Paulo, Dept Biochem, São Paulo, Brazil Univ Estadual Ponta Grossa, Dept Struct Mol Biol & Genet, Ponta Grossa, Brazil Catholic Univ Parana, Hlth & Biol Sci Inst, Curitiba, Parana, Brazil Universidade Federal de São Paulo, Dept Biochem, São Paulo, Brazil Web of Science

Bites from brown spiders (Loxosceles genus) have clinical manifestations including skin necrosis with gravitational spreading, and systemic involvement that may include renal failure, hemolysis, and thrombocytopenia. Mice were exposed to recombinant wild-type phospholipase-D, or to an isoform with a mutation in the catalytic domain resulting in no phospholipasic activity. Renal biopsies from mice treated with the wild-type toxin showed glomerular edema, erythrocytes and collapse of Bowman's space, edema and deposition of proteinaceous material within the tubular lumen. Ultrastructural analyses confirmed cytotoxicity by demonstrating disorders of glomerulus at foot processes and at fenestrated endothelium. Tubule alterations include deposits of amorphous material and edema, as well as an increase of epithelial cytoplasmic multivesicular bodies and electron-dense structures. There was an absence of nephrotoxicity in mice treated with the mutated toxin. Analyses of urine and blood showed that wild type toxin induced hematuria and elevation of blood urea, while treatment with mutated toxin caused no changes. Mouse lethality experiments also showed oliguria and mortality after treatment with wild-type toxin, but not following exposure to the mutated toxin. Immunofluorescence using antibodies to phospholipase-D toxin showed deposition of both toxins along the renal tubular structures as detected by confocal microscopy. Immunoblots of urine showed a 30 kDa band in samples from animals treated with wild-type toxin, but no band from mice exposed to mutated toxin. Wild-type toxin treatment caused cytoplasmic vacuolization, impaired spreading, reduction of cellular viability, and cell-cell and cell-substratum detachment in MDCK cells, while treatment with mutated isoform had no effect. Finally, there is a direct correlation between toxin activity on cell membrane phospholipids generating choline and cytotoxicity. We have defined for the first time a molecular mechanism for Loxosceles venom nephrotoxicity that is dependent on the catalytic activity of phospholipase-D toxin.

Loxoscelism is the designation given to clinical symptoms evoked by Loxosceles spider's bites. Clinical manifestations include skin necrosis with gravitational spreading and systemic disturbs. The venom contains several enzymatic toxins. Herein, we describe the cloning, expression, refolding and biological evaluation of a novel brown spider protein characterized as a hyaluronidase. Employing a venom gland cDNA library, we cloned a hyaluronidase (1200 bp cDNA) that encodes for a signal peptide and a mature protein. Amino acid alignment revealed a structural relationship with members of hyaluronidase family, such as scorpion and snake species. Recombinant hyaluronidase was expressed as N-terminal His-tag fusion protein (∼45 kDa) in inclusion bodies and activity was achieved using refolding. Immunoblot analysis showed that antibodies that recognize the recombinant protein cross-reacted with hyaluronidase from whole venom as well as an anti-venom serum reacted with recombinant protein. Recombinant hyaluronidase was able to degrade purified hyaluronic acid (HA) and chondroitin sulfate (CS), while dermatan sulfate (DS) and heparan sulfate (HS) were not affected. Zymograph experiments resulted in ∼45 kDa lytic zones in hyaluronic acid (HA) and chondroitin sulfate (CS) substrates. Through in vivo experiments of dermonecrosis using rabbit skin, the recombinant hyaluronidase was shown to increase the dermonecrotic effect produced by recombinant dermonecrotic toxin from L. intermedia venom (LiRecDT1). These data support the hypothesis that hyaluronidase is a “spreading factor”. Recombinant hyaluronidase provides a useful tool for biotechnological ends. We propose the name Dietrich's Hyaluronidase for this enzyme, in honor of Professor Carl Peter von Dietrich, who dedicated his life to studying proteoglycans and glycosaminoglycans., Author Summary Accidents involving brown spiders (Loxosceles genus) are reported throughout the world. South and Southeast of Brazil are endemic areas for this spider. Loxosceles bites commonly trigger local signs as swelling, erythema, hemorrhage and the hallmark symptom: a dermonecrotic lesion with gravitational spreading. Systemic effects are less common; however, are implicated in more severe cases. Hyaluronidases are referred in several venoms as “spreading factors” due to their enzymatic activity upon extracellular components. This activity facilitates the permeation of other toxins through the victim's body. In fact, a previous study identified the activity of L. intermedia venom upon glycosaminoglycans which are abundant components in the extracellular matrix of many tissues. Disclosing a little more about the role of hyaluronidases within this venom, we investigated the activities of a recombinant hyaluronidase from L. intermedia venom. Dietrich's hyaluronidase, as it was designated, was produced as a recombinant protein. By performing a rabbit skin dermonecrosis assay using Dietrich's Hyaluronidase and a dermonecrotic toxin, we showed that Dietrich's Hyaluronidase increased the dermonecrotic area induced by the dermonecrotic toxin. Our results confirm that hyaluronidases are a “spreading factor” of L. intermedia venom.

Spiders of the Loxosceles genus are cosmopolitan, and their venom components possess remarkable biological properties associated with their ability to act upon different molecules and receptors. Accidents with Loxosceles intermedia specimens are recognized as a public health problem in the south of Brazil. To describe the transcriptional profile of the L. intermedia venom gland, we generated a wide cDNA library, and its transcripts were functionally and structurally analyzed. After initial analyses, 1843 expressed sequence tags (ESTs) produced readable sequences that were grouped into 538 clusters, 281 of which were singletons. 985 reads (53% of total ESTs) matched to known proteins. Similarity searches showed that toxin-encoding transcripts account for 43% of the total library and comprise a great number of ESTs. The most frequent toxins were from the LiTx family, which are known for their insecticidal activity. Both phospholipase D and astacin-like metalloproteases toxins account for approximately 9% of total transcripts. Toxins components such as serine proteases, hyaluronidases and venom allergens were also found but with minor representation. Almost 10% of the ESTs encode for proteins involved in cellular processes. These data provide an important overview of the L. intermedia venom gland expression scenario and revealed significant differences from profiles of other spiders from the Loxosceles genus. Furthermore, our results also confirm that this venom constitutes an amazing source of novel compounds with potential agrochemical, industrial and pharmacological applications.