Background Developing effective vaccines against SARS-CoV-2 that consider manufacturing limitations, equitable access, and acceptance is necessary for developing platforms to produce antigens that can be efficiently presented for generating neutralizing antibodies and as a model for new vaccines. Results This work presents the development of an applicable technology through the oral administration of the SARS-CoV-2 RBD antigen fused with a peptide to improve its antigenic presentation. We focused on the development and production of the recombinant receptor binding domain (RBD) produced in E. coli modified with the addition of amino acids extension designed to improve antigen presentation. The production was carried out in shake flask and bioreactor cultures, obtaining around 200 mg/L of the antigen. The peptide-fused RBD and peptide-free RBD proteins were characterized and compared using SDS-PAGE gel, high-performance chromatography, and circular dichroism. The peptide-fused RBD was formulated in an oil-in-water emulsion for oral mice immunization. The peptide-fused RBD, compared to RBD, induced robust IgG production in mice, capable of recognizing the recombinant RBD in Enzyme-linked immunosorbent assays. In addition, the peptide-fused RBD generated neutralizing antibodies in the sera of the dosed mice. The formulation showed no reactive episodes and no changes in temperature or vomiting. Conclusions Our study demonstrated the effectiveness of the designed peptide added to the RBD to improve antigen immunostimulation by oral administration. Graphical Abstract

Proteomic characterization of Micrurus browni browni venom showed approximately 41 components belonging to 9 protein families, mainly phospholipases A(2) (PLA(2)s) and three-finger toxins (3FTxs). Venom gland transcriptome yielded 39 venom transcripts belonging to 10 protein families. Functional characterization identified a multimeric toxin, here designated Brownitoxin-1, which comprises at least one PLA(2) and one 3FTx. Its components have no or very low lethality individually but become extremely lethal when combined; both were partially characterized. Other two lethal components were identified: A neurotoxic PLA(2), and a postsynaptic alpha-neurotoxin. LD(50)s as well as PLA(2) and nAChR-blocking activities were determined for whole venom and isolated components. Application of venom to murine neuromuscular preparations caused a progressive decrease of twitch force that was irreversible after washing. Inhibition of PLA(2) activity with p-bromophenacyl bromide (pBPB) showed that approximately 90% of toxicity is dependent on this activity. Non-lethal components include diverse 3FTxs, at least three enzymatically active PLA(2)s and the nociceptive toxin MitTx. No evidence of specificity towards prey was observed. This work is one of the most complete characterizations of a coral snake venom so far and its findings highlight the relevance of protein complexes in venom function. Significance: This study represents a profound analysis of the venom of the coral snake Micrurus browni browni, including a venom proteome, venom gland transcriptomic data and functional studies of whole venom and isolated toxins. It significantly contributes to the understanding of North American coral snake venoms, which are currently largely unknown. It includes characterization of relevant venom components, one of which represents the first description of a lethal multimeric neurotoxin in coral snake venom. This work highlights the importance of protein complexes in coral snake venom and could serve as a basis for the finding of several other multimeric toxins. Finally, we report the absence of taxon specificity, which has been previously reported in the venoms of other snakes of the same genus.

The most abundant protein families in viper venoms are Snake Venom Metalloproteases (SVMPs), Snake Venom Serine Proteases (SVSPs) and Phospholipases (PLA2s). These are primarily responsible for the pathophysiology caused by the bite of pit-vipers; however, there are few studies that analyze the pharmacokinetics (PK) of whole venom (WV) and its protein families. We studied the pathophysiology, PK profile and differential absorption of representative toxins from venom of Neotropical Rattlesnake (Crotalus simus) in a large animal model (ovine). Toxins studied included crotoxin (the main lethal component), which causes moderate to severe neurotoxicity; SVSPs, which deplete fibrinogen; and SVMPs, which cause local tissue damage and local and systemic hemorrhage. We found that Whole Venom (WV) was highly bioavailable (86%) 60 h following intramuscular (IM) injection, and extrapolation suggests that bioavailability may be as high as 92%. PK profiles of individual toxins were consistent with their physicochemical properties and expected clinical effects. Lymph cannulated animals absorbed 1.9% of WV through lymph during the first 12 h. Crotoxin was minimally detectable in serum after intravenous (IV) injection; however, following IM injection it was detected in lymph but not in blood. This suggests that crotoxin is quickly released from the blood toward its tissue targets.

e13099 Background: Pregnancy-associated breast cancer (PABC) includes breast cancer diagnosed during pregnancy or within one or two years after delivery. PABC is the second most common malignancy diagnosed in pregnancy. There is controversial information about outcomes in this setting. Methods: We conducted a retrospective case-control study to evaluate overall survival (OS) and progresion free survival (PFS) of pregnant versus non-pregnant women with breast cancer, matched for age, inmunohistochemical subtype and stage of disease. Results: Median age was 35 years, there were no differences between cases and controls regarding clinical stage, tumor grading, IHC subtype (p = 1.0), Ki67 and histological type. Highlight that 31% were triple negative. Cases and controls were followed over 48 months. Kaplan–Meier survival showed no significant difference in OS and PFS between cases and controls (p = 0.892 and 0.121 respectivily). But if we analized the population by the time breast cancer takes place, the percentage of patients free of progresión at 24 months were: during pregnancy 46.4 (CI 95% 27.9-64.8); 12 months-postpartum 29.6 (CI 95% 12.3-46.8) and non-pregnancy patients 26.7 (CI 95% 17.2-36.1), log-rank test showed significance difference between these population PFS curves p = 0.045. Conclusions: We confirm previous reports that patients with breast cancer after delivery had worst PFS and PABC it is not associated with poor OS. As far as we know this is the first hispanic report in this setting. PABC %(n/N) Non-PABC %(n/N) P Clinical stage 1.0 I 1.7 (2/116) 1.7 (4/232) II 29.3 (34/116) 29.3 (68/232) III 48.3 (56/116) 48.3 (112/232) IV 20.7 (24/116) 20.7 (48/232) Histological type n (%) 0.171 Canalicular carcinoma 76.7 (89/116) 85.3 (198/232) Lubulillar carcinoma 12.1 (14/116) 6.0 (14/232) Mix 6.0 (7/116) 3.9 (9/232) Other 5.2 (6/116) 4.7 (11/232) Subtype 1.0 Triple Negative 31.0 (36/116) 31.0 (72/232) HER2+ 27.6 (32/116) 27.6 (64/232) RH+ 41.4 (48/116) 41.4 (96/232) Ki-67 n (%) 0.470 < 20 45.8 (44/96) 44.6 (100/224) > 20 54.2 (52/96) 55.4 (124/224) SBR CCI y CLI n (%)* 0.802 Low 9.1 (10/110) 8.1 (18/221) Medium 37.4 (41/110) 34.4 (76/221) High 53.6 (59/110) 57.5 (127/221)

Shake flasks are widely used during the development of bioprocesses for recombinant proteins. Cultures of recombinant Escherichia coli with orbital mixing (OM) have an oxygen limitation negatively affecting biomass growth and recombinant-protein production. With the aim to improve mixing and aeration in shake flask cultures, we analyzed cultures subjected to OM and the novel resonant acoustic mixing (RAM) by applying acoustic energy to E. coli BL21-Gold (DE3): a producer of recombinant phospholipase A2 (rPLA2) from Micrurus laticollaris snake venom.

Aggregation of recombinant proteins into inclusion bodies (IBs) is the major drawback of heterologous expression in Escherichia coli. Here, we evaluated the effects of a pH shift after expression induction on recombinant phospholipase A2 production and its aggregation in IBs in E. coli Origami™, as compared to cultures with pH maintained at 7.5 or uncontrolled pH. Cultures shifted from 7.5 to pH 6.5 or 8.5 produced ∼15–25% less biomass as compared with those kept at 7.5 or without pH control. The cultures shifted to pH 8.5 showed a ∼50% higher yield of acetate per biomass, and the rPLA2 yield was improved 2.4-fold. Purified IBs formed at pH 8.5 containing ∼50% of rPLA2, were more susceptible to proteinase-K cleavage and bound less thioflavin-T, indicating lower amyloid content, with the concomitant enrichment of α-helical and random-coil secondary structures, as demonstrated by FTIR. Moreover, only one IB per cell was formed at pH 8.5; instead, more than two were observed under the other culture pH conditions. Nevertheless, under uncontrolled pH conditions, ∼300nm larger IBs were observed. Our work presents evidence of the usefulness of recombinant protein expression cultivated at pH 8.5 allowing the reduction of amyloid content in IBs.

This work reports a new approach to clone active Neopladine 2 (Neo2), an anticancer peptide from Tityus discrepans scorpion venom. RNA was extracted from the venom gland and cDNA was obtained by RTPCR using complementary primers for the nucleotide sequence coding native Neo2 (nNeo2) N-terminal. Deduced amino acid sequence shows that Neo2 is comprises by 245 residues. The best conditions to achieve active Neo2 expression in Escherichia coli were 16 degrees C for 20-24 h. Active recombinant Neo2 (rNeo2) was obtained as inclusion bodies (IBs) with a yield of 3.5 mg/L culture. rNeo2 purification by HPLC revealed eleven isoforms with differences in polarity and activity. Only rNeo2a and rNeo2k isoforms induces cancer cells death like nNeo2. rNeo2a, rNeo2g and rNeo2j were the most interesting isoforms because their apoptosis/necrosis (A/N) index and their yield. Cloned isoforms have attractive properties because some of them had higher activity than the native protein, which can be exploited for biotechnological and biomedical applications. The in silico Neo2 3D structure predicted a globular protein, with five alpha-helixes and four beta-sheet maintained by 4 sulfide pairing, highly polar, with charged amino acids in its surface and two faces with different electrostatic potentials, suggesting an important dipolar moment. (C) 2016 Published by Elsevier Ltd.

Background Inclusion bodies (IBs) are aggregated proteins that form clusters when protein is overexpressed in heterologous expression systems. IBs have been considered as non-usable proteins, but recently they are being used as functional materials, catalytic particles, drug delivery agents, immunogenic structures, and as a raw material in recombinant therapeutic protein purification. However, few studies have been made to understand how culture conditions affect the protein aggregation and the physicochemical characteristics that lead them to cluster. The objective of our research was to understand how pH affects the physicochemical properties of IBs formed by the recombinant sphingomyelinase-D of tick expressed in E. coli BL21-Gold (DE3) by evaluating two pH culture strategies. Results Uncontrolled pH culture conditions favored recombinant sphingomyelinase-D aggregation and IB formation. The IBs of sphingomyelinase-D produced under controlled pH at 7.5 and after 24 h were smaller (<500 nm) than those produced under uncontrolled pH conditions (>500 nm). Furthermore, the composition, conformation and β-structure formation of the aggregates were different. Under controlled pH conditions in comparison to uncontrolled conditions, the produced IBs presented higher resistance to denaturants and proteinase-K degradation, presented β-structure, but apparently as time passes the IBs become compacted and less sensitive to amyloid dye binding. Conclusions The manipulation of the pH has an impact on IB formation and their physicochemical characteristics. Particularly, uncontrolled pH conditions favored the protein aggregation and sphingomyelinase-D IB formation. The evidence may lead to find methodologies for bioprocesses to obtain biomaterials with particular characteristics, extending the application possibilities of the inclusion bodies.

Corrigendum to “Intraspecific differences in the immunochemical reactivity and neutralization of venom from Argentinean Bothrops (Rhinocerophis) alternatus by specific experimental antivenoms” [Toxicon 85 (2014) 31e45] Laura Cecilia Lanari , Alejandro Olvera , Vanessa Costa de Oliveira , Rodrigo Daniel Laskowicz , Leslie Boyer , N estor Rub en Lago , Alejandro Alag on , Adolfo Rafael de Roodt a, c, * a National Institute for Production of Biologicals, Ministry of Health, Buenos Aires, Argentina b Institute of Biotechnology, National Autonomous University of Mexico, Cuernavaca, Morelos, Mexico c Laboratory of Toxinopathology, Center of Experimental and Applied Pathology, Faculty of Medicine, University of Buenos Aires, Buenos Aires, Argentina d VIPER Institute, University of Arizona, Tucson, AZ, USA

The venoms of Bothrops (Rhinocerophis) alternatus (B.a.) from different regions of Argentina have shown biochemical, toxicological and immunological variations. Considering these variations, we produced nine experimental antisera (rabbit, IgG) against venoms from snakes of nine different regions and a pool of venom, comprised of equal amounts of venoms from each region. The immunologic studies (ELISA, Westernblot) showed significant cross reactivity among all regional antivenoms with all regional venoms, with no significant differences regarding the specificity of the immunogens used for the production of antivenom. Neutralization of hemorrhage was variable (although all the antivenoms neutralized this activity in all venoms) and the neutralization of coagulant and phospholipase activities were evident in all cases. Some antivenoms neutralized toxic activities that were absent or very low in the venoms used as immunogen, on other non-homologous venoms (e.g. thrombin like activity). Despite the different toxic potencies of regional venoms, antivenoms developed using venoms of snakes from a particular region showed high immunochemical reactivity and cross-neutralizing capacity on snake venoms from different and distant regions, in occasions over those of the homologous antivenoms. These findings could be used to improve the generation of pools of venoms for the production of antivenoms.

Hyaluronidases (Hyal) present in the venom of poisonous animals have been considered as “spreading factors” that facilitate a fast penetration of the venom in the prey. We have found that hyaluronidase from the tarantula Brachypelma vagans venom (BvHyal) displays a substrate-specific Hyal activity against hyaluronan. By using a combined strategy based on peptide sequencing and RT-PCR, we have cloned a BvHyal cDNA. Active recombinant BvHyal was efficiently expressed in a baculovirus system in insect cell.

epidermis TO THE EDITOR We thank the authors for presenting these valuable comments and for giving us the opportunity to extend the discussion limited by the format of the paper. van Meeteren et al., 2004van Meeteren L.A. Frederiks F. Giepmans B.N. Pedrosa M.F. Billington S.J. Jost B.H. et al.Sphingomyelinases D target cellular lysophosphatidic acid receptors by hydrolyzing lysophosphatidylcholine.J Biol Chem. 2004; 27: 10833-10836Google Scholar confirmed that recombinant SMDs from Loxosceles laeta and Corynebacterium pseudotuberculosis possessed intrinsic lysophospholipase D activity to generate bioactive lysophosphatidic acid. They demonstrated that SMD did not activate mitogen-activated protein kinase (ERK1/2) in receptor-deficient B103 neuroblastoma cells, whereas both SMDs were activating mitogen-activated protein kinase when the same cells expressed lysophosphatidic acid1 receptors. They also showed that bacterial and Loxosceles SMDs triggered receptor internalization in HEK293 cells only when preincubated with albumin-LPC. Lee and Lynch, 2005Lee S. Lynch K.R. Brown recluse spider (Loxosceles reclusa) venom phospholipase D (PLD) generates lysophosphatidic acid (LPA).Biochem J. 2005; 391: 317-323Google Scholar extended the findings by demonstrating that recombinant L. reclusa SMD hydrolzses various lysophospholipids and identified specific histidine residues that are essential for the enzyme activity. Pettus et al., 2003Pettus B.J. Bielawska A. Spiegel S. Roddy P. Hannun Y.A. Chalfant C.E. Ceramide kinase mediates cytokine- and calcium ionophore-induced arachidonic acid release.J Biol Chem. 2003; 278: 38206-38213Google Scholar, Pettus et al., 2004Pettus B.J. Bielawska A. Subramanian P. Wijesinghe D.S. Maceyka M. Leslie C.C. et al.Ceramide 1-phosphate is a direct activator of cytosolic phospholipase A2.J Biol Chem. 2004; 27: 11320-11326Google Scholar in two consequential investigations provided evidence that ceramide-1-phosphate (C1P) interacts directly with cytosolic phospholipase A2 (cPLA2) acting as an activator of cPLA2 and subsequent inflammatory response. They determined that in A549 lung adenocarcinoma cells, natural and endogenous (produced by SMD) C1Ps were potent and specific inducers of arachidonic acid and prostanoid synthesis. The treatment of A549 cells with SMD resulted in a threefold increase in arachidonic acid release. Using RNA-mediated interference technology, Pettus et al. confirmed in A549 and in J774.1 microphages that cPLA2 was downstream of C1P. It was also found that in A549 cells, C1P caused translocation of cPLA2 to membranes. Their in vitro binding studies disclosed that C1P directly binds and activate with full-length cPLA2. Other publications regarding biological activities of C1P are reviewed in details by Gomez-Munoz, 2004Gomez-Munoz A. Ceramide-1-phosphate: a novel regulator of cell activation.FEBS Lett. 2004; 562: 5-10Google Scholar. The focus of our investigation was the expression pattern of human fibroblasts treated with recombinant SMD to gain insight into cellular mechanisms of loxoscelism pathology. We observed a dose- and time-dependent upregulation of several cytokines (data not shown). The pattern of continuous increase was present up to 18 hours of treatment (data not shown). Although we recognize the evidence that lysophospholipase D activity of SMD is obviously an important factor in loxoscelism, we are not convinced that the sphingomyelin– ceramide pathway involvement in the L. reclusa pathology can be ignored. More than one mechanism of action is also possible in this pathology and further investigation would benefit the understanding of the complex immunological response following spider envenomation. The authors state no conflict of interest.

The toxic, biochemical, and immunological characteristics of L. boneti and L. reclusa venoms and its neutralization by anti-L. boneti and anti-L. reclusa antivenoms were studied. The electrophoretic profile showed very similar patterns and the toxic activities were very close. Immunological studies showed cross-reactivity among L. boneti and L. reclusa venoms, with L. boneti and L. reclusa experimental antivenoms, and anti-L. gaucho and anti-L. laeta antivenoms. The venom of L. laeta showed low immunological reactivity with the North American Loxosceles antivenoms. Experimental anti-North American Loxosceles antivenoms protected mice of the systemic toxicity and were able to prevent necrosis in rabbit skin after the injection of the venom. Both antivenoms displayed cross neutralization. The results showed that both Loxosceles venoms have very close toxic, biochemical, and immunological characteristics, and that either monospecific antivenoms or an antivenom raised with L. boneti and L. reclusa venoms as immunogens could be useful for treating bites by North American Loxosceles spiders.

sphingomyelinase D lysophosphatidic acid ceramide-1-phosphate TO THE EDITOR In a recent report, Dragulev et al., 2006Dragulev B. Bao Y. Ramos-Cerrillo B. Vazquez H. Olvera A. Stock R. et al.Upregulation of IL-6, IL-8, CXCL1, and CXCL2 dominates gene expression in human fibroblast cells exposed to Loxosceles reclusa sphingomyelinase D: insights into spider venom dermonecrosis.J Invest Dermatol. 2006https://doi.org/10.1038/sj.jid.5700644Google Scholar tested the hypothesis that stromal fibroblasts may participate in the pathophysiology of Loxosceles spider envenomation, which is characterized by local skin injury as well as systemic toxicity including severe inflammation. To this end, the authors determined the fibroblast gene expression response to sphingomyelinase D (SMD), the causative agent of Loxosceles venom. They found that SMD upregulates the expression of proinflammatory cytokines and chemokines, such as IL-6, IL-8, CXCL1, and CXCL2. On the basis of these results, the authors hypothesized that ceramide-1-phosphate (C1P), the product of sphingomyelin (SM) hydrolysis in the outer leaflet of the plasma membrane, is responsible for the observed upregulation of proinflammatory genes. However, although the conversion of SM to C1P in the outer leaflet may modulate ion channel activity (Ramu et al., 2006Ramu Y. Xu Y. Lu Z. Enzymatic activation of voltage-gated potassium channels.Nature. 2006; 442: 696-699Google Scholar), C1P is not obviously a signaling molecule. How then does SMD trigger an inflammatory response in its target cells? What the authors do not mention is the fact that SMD has intrinsic lysophospholipase D activity to generate the lipid mediator lysophosphatidic acid (LPA) from lysophosphatidylcholine (van Meeteren et al., 2004van Meeteren L.A. Frederiks F. Giepmans B.N. Pedrosa M.F. Billington S.J. Jost B.H. et al.Spider and bacterial sphingomyelinases D target cellular lysophosphatidic acid receptors by hydrolyzing lysophosphatidylcholine.J Biol Chem. 2004; 279: 10833-10836Google Scholar; Lee and Lynch, 2005Lee S. Lynch K.R. Brown recluse spider (Loxosceles reclusa) venom phospholipase D (PLD) generates lysophosphatidic acid (LPA).Biochem J. 2005; 391: 317-323Google Scholar). Through activation of its cognate G protein-coupled receptors, LPA exerts numerous biological and pathophysiological responses in many different cell types (Moolenaar et al., 2004Moolenaar W.H. van Meeteren L.A. Giepmans B.N. The ins and outs of lysophosphatidic acid signaling.Bioessays. 2004; 26: 870-881Google Scholar). By producing bioactive LPA, SMD can activate LPA receptor-mediated signaling pathways that may impinge on inflammatory gene expression; indeed, LPA induces the expression of various cytokines, including IL-6, IL-8, CXCL1, and CCL2 (e.g., Palmetshofer et al., 1999Palmetshofer A. Robson S.C. Nehls V. Lysophosphatidic acid activates nuclear factor kappa B and induces proinflammatory gene expression in endothelial cells.Thromb Haemost. 1999; 82: 1532-1537Google Scholar; Fang et al., 2004Fang X. Yu S. Bast R.C. Liu S. Xu H.J. Hu S.X. et al.Mechanisms for lysophosphatidic acid-induced cytokine production in ovarian cancer cells.J Biol Chem. 2004; 279: 9653-9661Google Scholar; Klemm et al., 2007Klemm S. Zimmermann S. Peschel C. Mak T.W. Ruland J. Bcl10 and Malt1 control lysophosphatidic acid-induced NF(kappa)B activation and cytokine production.Proc Natl Acad Sci USA. 2007; 104: 134-138Google Scholar; C. Stortelers, unpublished results). That LPA is a key mediator of SMD activity is demonstrated by the failure of SMD to evoke biological effects in LPA receptor-negative cells (van Meeteren et al., 2004van Meeteren L.A. Frederiks F. Giepmans B.N. Pedrosa M.F. Billington S.J. Jost B.H. et al.Spider and bacterial sphingomyelinases D target cellular lysophosphatidic acid receptors by hydrolyzing lysophosphatidylcholine.J Biol Chem. 2004; 279: 10833-10836Google Scholar). Thus, LPA rather than C1P is the likely trigger of the observed inflammatory response to Loxosceles SMD. Specific LPA antagonists could be useful tools for the treatment of bites by Loxosceles spiders. The authors state no conflict of interest.

sphingomyelinase D TO THE EDITOR Brown recluse (Loxosceles spp.) spiders are arachnid species known to cause necrotic arachnidism. The envenomation, described as loxoscelism, is associated with localized pain, erythema, and edema followed by the development of necrosis (Futrell, 1992Futrell J. Loxoscelism.Am J Med Sci. 1992; 304: 261-267Google Scholar; Hogan et al., 2004Hogan C. Barbaro K. Winkel K. Loxoscelism: old obstacles, new directions.Ann Emerg Med. 2004; 44: 608-624Google Scholar). However, the specific pathophysiological mechanisms by which Loxosceles venom exerts these noxious symptoms are multifactorial and not fully understood. The causative factor for production of necrotic lesions is generally considered to be the enzyme sphingomyelinase D (SMD), which cleaves sphingomyelin to form choline and ceramide 1-phosphate. Four active forms of SMD with molecular mass of 32,000 are found in L. recluse (Kurpiewski et al., 1981Kurpiewski G. Forrester L.J. Barrett J.T. Campbell J. Platelet aggregation and sphingomyelinase D activity of a purified toxin from the venom of Loxosceles reclusa.Biochim Biophys Acta. 1981; 678: 467-476Google Scholar; Futrell, 1992Futrell J. Loxoscelism.Am J Med Sci. 1992; 304: 261-267Google Scholar). Sphingomyelinases of comparable size are also described in the venoms of L. intermedia, L. gaucho, and L. laeta (Silva et al., 2004Silva P.H. Silveira R.B. Appel M.H. Mangili O.C. Gremski W. Veiga S.S. Brown spiders and loxoscelism.Toxicon. 2004; 44: 693-709Google Scholar). Pedrosa et al., 2002Pedrosa M.F.F. Azevedo I.L.M.J. Goncalves-de-Andrade R.M. van den Berg C.W. Ramos C.R.R. Ho P.L. et al.Molecular cloning and expression of a functional dermonecrotic and haemolytic factor from Loxosceles laeta venom.Biochem Biophys Res Commun. 2002; 298: 638-645Google Scholar reported that initial incubation with specific antiserum of biologically functional recombinant L. laeta SMD inhibited dermonecrotic activity. The fact that SMD enzymatic activity is necessary for initiation of the necrosis was confirmed by Ramos-Cerrillo et al., 2004Ramos-Cerrillo B. Olvera A. Odell G. Zamudio F. Paniagua-Solís J. Alagón A. et al.Genetic and enzymatic characterization of sphingomyelinase D isoforms from the North American fiddleback spiders Loxosceles boneti and Loxosceles recluse.Toxicon. 2004; 44: 507-514Google Scholar. They analyzed three recombinant L. boneti SMD isoforms for enzymatic activity in vitro, and dermonecrosis when injected in rabbits. Isoforms 1 and 2 retained enzymatic activity and caused dermonecrosis in vivo. Interestingly, isoform 3 which did not produce dermonecrosis was also enzymatically inactive. Similar results for SMD isoforms from L. intermedia were previously reported by Tambourgi et al., 1998Tambourgi D.V. Magnoli F.C. van den Berg C.W. Morgan B.P. de Araujo P.S. Alves E.W. et al.Sphingomyelinases in the venom of the spider Loxosceles intermedia are responsible for both dermonecrosis and complement dependent hemolysis.Biochem Biophys Res Commun. 1998; 251: 366-373Google Scholar. It is currently thought that SMD does not directly induce necrosis but rather it acts as initiator of the pathology by stimulating inflammatory response in endothelial cells (Patel et al., 1994Patel K.D. Modur V. Zimmerman G.A. The necrotic venom of the brown recluse spider induces dysregulated endothelial cell-dependent neutrophil activation: differential induction of GM-CSF, IL-8, and E-selectin expression.J Clin Invest. 1994; 94: 631-642Google Scholar; Desai et al., 1999Desai A. Miller M.J. Gomez H.F. Warren J.S. Loxosceles deserta spider venom induces NF-κB-dependent chemokine production by endothelial cells.Clin Toxicol. 1999; 37: 447-456Google Scholar, Desai et al., 2000Desai A. Lankford H.A. Warren J.S. Loxosceles deserta spider venom induces the expression of vascular endothelial growth factor (VEGF) in keratinocytes.Inflammation. 2000; 24: 1-9Google Scholar). We hypothesize that the stromal fibroblasts are also involved in loxosceles pathophysiology. To test this as well as further investigate in vitro the mechanisms associated with loxoscelism, we assessed the gene expression profiles of SMD-treated and untreated human fibroblasts using GeneChips, protein microarrays, and quantitative reverse transcription-PCR. Human fibroblasts cells (ATCC CRL-1635) maintained in DMEM, containing 10% serum were used in these experiments. At the time of treatment, the media was replaced with serum-free DMEM. After 4 hours of incubation the media was changed and the cells were cultured with 1 μg/ml L. recluse isoform I recombinant SMD (Olvera et al., 2006Olvera A. Ramos-Cerrillo B. Estevez J. Clement H. Roodt A. Paniagua-Solıs J. et al.North and south American Loxosceles spiders: development of a polyvalent antivenom with recombinant sphingomyelinases D as antigens.Toxicon. 2006; 48: 64-74Google Scholar) in DMEM without serum for 30 minutes, 1, 3, or 5 hours. At this concentration of toxin, there was no decernable cell injury, and greater than 95% of cells remained viable (by trypan blue exclusion assay) after 48 hours of incubation. This concentration for the SDM toxin was chosen based on a previously report (Gomez et al., 2001Gomez H.F. Greenfield D.M. Miller M.J. Warren J.S. Direct correlation between diffusion of Loxosceles recluse and the extent of dermal inflammation.Acad Emerg Med. 2001; 8: 309-314Google Scholar) where an intradermal application of 3 μg complete L. recluse venom showed reproducible pathology in the rabbit model of envenomation. Using AMPLEX assay (Invitrogen, Carlsbad, CA), we found that of 1 μg of SMD showed sphingomyelinase activity corresponding to the enzyme activity of 3 μg of our venom preparation. All experiments were approved by the Institutional Biosafety Committee of the University of Virginia. The gene expression profiles of two samples after 5 hours of SMD treatment were compared with two controls using Affymetrix Hg-U133A arrays. To identify the differentially expressed genes between treated and the control samples chip data were analyzed using the Affymetrix Data Mining Tool. Similar experiments were performed using whole L. recluse venom (5 μg/ml) and comparable changes in expression were observed as to those in with the SDM toxin (data not shown) suggesting that the SDM toxin is the primary contributor to changes in fibroblast gene expression. Three groups of upregulated genes from the SDM-treated fibroblast gene expression experiments were considered of further interest in relation to the molecular mechanisms associated with loxosceles pathology: human cytokines (IL-6, IL-8, IL-1B, CXCL1, CXCL2, CCL5, tumor necrosis factor); genes involved in glycosphingolipid metabolism pathway (alpha 1,4-galactosyltransferase, UDP-glucose ceramide glucosyltransferase); and proteins known as factors of transcriptional regulation (nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 (p49/p100), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilon). The expression profiles of seven arbitrarily selected regulated cytokines and NF-κB were validated by qPCR (Table 1). To determine if the alterations in transcription actually lead to increase protein expression media from treated and untreated fibroblasts (5 hours) were analyzed with FASTQuant Human II protein microarrays (Table 1). To extend the analysis of the upregulated cytokines, expression levels at four time points following treatment were investigated. Changes were first observed at 3 hours following SMD treatment, and at 5 hours significant upregulation of IL-6, IL-8, CXCL-1, and CXCL-2 was observed (Figure 1).Table 1Gene expression and protein fold changes of selected genes in SMD-treated fibroblast culturesGene nameFold changeGeneChip1The microarray data (including the chp, cel, dat, and txt files) are available and publicly accessible at https://genes.med.virginia.edu/public_files/Jay_Fox/Jay_Fox_Subtoxic%20Effects%20of%20Snake%20Venoms%20on%20HUVEC_all.zip. The matrix of criteria employed for analysis was: fold changes ≥1.5; T-test P-value ≤0.05; signal difference=Min (100, 5 × 20th percentile of baseline noise); detection calls P%=50 for upregulated genes, or <50 for downregulated genes.qRT-PCR2The fold change values presented as mean±SD were calculated using the relative quantification method (RQ=2−ΔΔCt); Applied Biosystems, Foster City, CA (2001). Human glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene for normalization.Protein array3The data presented correspond to mean value±SD of three technical replicates. Standard curves were generated using logistic (ELISA) settings for each cytokine and the protein concentrations were calculated using ArrayVision FAST software (Whatman Inc., Florham Park, NJ).IL-616.1927.2±2.2660.72±3.28IL-8193.62722±63.9841.43±5.35IL-1B6.1410.0±0.161.15±0.14CXCL164.87277.65±5.45Not analyzedCXCL2130.46226.03.0±18.95Not analyzedCCL53.5757.72±1.845.46±2.62TNF-α5.598.04±0.43Not analyzedNF-κB2.534.10±0.06Not analyzedqRT-PCR, quantitative reverse transcriptase-PCR; SMD, sphingomyelinase D; TNF-α, tumor necrosis factor.1 The microarray data (including the chp, cel, dat, and txt files) are available and publicly accessible at https://genes.med.virginia.edu/public_files/Jay_Fox/Jay_Fox_Subtoxic%20Effects%20of%20Snake%20Venoms%20on%20HUVEC_all.zip. The matrix of criteria employed for analysis was: fold changes ≥1.5; T-test P-value ≤0.05; signal difference=Min (100, 5 × 20th percentile of baseline noise); detection calls P%=50 for upregulated genes, or <50 for downregulated genes.2 The fold change values presented as mean±SD were calculated using the relative quantification method (RQ=2−ΔΔCt); Applied Biosystems, Foster City, CA (2001). Human glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene for normalization.3 The data presented correspond to mean value±SD of three technical replicates. Standard curves were generated using logistic (ELISA) settings for each cytokine and the protein concentrations were calculated using ArrayVision FAST software (Whatman Inc., Florham Park, NJ). Open table in a new tab qRT-PCR, quantitative reverse transcriptase-PCR; SMD, sphingomyelinase D; TNF-α, tumor necrosis factor. In summary our study focused on the global gene expression changes in human fibroblast cells, following incubation with recombinant SMD. We detected induction of a strong inflammatory response involving transcriptional upregulation of IL-6, IL-8, IL-1B, CCL5, CXCL1, CXCL2, and tumor necrosis factor-α in conjunction with a proportional increase in the expression of some of these proteins. We also observed a modest increase in the transcription of two genes involved in glycosphingolipid metabolism pathway (UDP-glucose ceramide glucosyltransferase and α-1,4-galactosyltransferase), as well as NF-κB and NFKBIA (NF-κB inhibitor). In contrast to a previous study conducted on endothelial cells (Patel et al., 1994Patel K.D. Modur V. Zimmerman G.A. The necrotic venom of the brown recluse spider induces dysregulated endothelial cell-dependent neutrophil activation: differential induction of GM-CSF, IL-8, and E-selectin expression.J Clin Invest. 1994; 94: 631-642Google Scholar), we found IL-6 significantly upregulated in fibroblast cultures treated with SMD. IL-6 and IL-8 expression is known to be activated via the transcriptional factor NF-κB (Matsusaka et al., 1993Matsusaka T. Fujikawa K. Nishio Y. Mukaida N. Matsushima K. Kishimoto T. et al.Transcription factors NF-IL6 and NF-κB synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8.Proc Natl Acad Sci USA. 1993; 90: 10193-10197Google Scholar). It is also noted in the literature (Bauerle and Henkel, 1994Bauerle P.A. Henkel T. Function and activation of NF-κB in immune system.Annu Rev Immunol. 1994; 12: 141-179Google Scholar; Ballou et al., 1996Ballou L. Laulederkind S. Rosloniec E. Raghow R. Ceramide signaling and the immune response.Biochim Biophys Acta. 1996; 1301: 273-287Google Scholar) that phosphorylated ceramide is an important factor in the activation of the transcriptional factor NF-κB. In the normal acute inflammatory process, neutrophils are the first cells recruited by the immune system, followed by their clearance and mononuclear cells infiltration. IL-6 is one of the regulators of this immunological switch (Kaplanski et al., 2003Kaplanski G. Marin V. Montero-Julian F. Alberto Mantovani A. Farnarier C. IL-6: a regulator of the transition from neutrophil to monocyte recruitment during inflammation.Trends Immunol. 2003; 24: 25-29Google Scholar; Jones, 2005Jones S. Directing transition from innate to acquired immunity: defining a role for IL-6.J Immunol. 2005; 175: 3463-3468Google Scholar). Thus, based on the results presented and the literature we speculate that SMD hydrolyzes sphingomyelins localized in the outer leaflet of the plasma membrane of fibroblasts and ceramide phosphate, which is the product of sphingomyelin hydrolysis, is responsible for an abnormal activation of NF-κB. Furthermore, we suggest that secretion of ceramide phosphate-induced IL-8 by endothelial cells and stromal fibroblasts in addition to the normally immunoregulated IL-8, might be connected to loxoscelism histopathology dominated, by dense neutrophilic infiltrate, necrosis, edema, and proliferation of fibroblasts (Elston et al., 2000Elston D. Eggers J. Schmidt W. Storrow A. Doe R. McGlasson D. et al.Histological findings after brown recluse spider envenomation.Am J Dermatopathol. 2000; 22: 242-246Google Scholar; Tambourgi et al., 2005Tambourgi D.V. Paixao-Cavalcante D. Goncalves de Andrade R.M. Fernandes-Pedrosa M.F. Magnoli F.C. Morgan B.P. et al.Loxosceles sphingomyelinase induces complement-dependent dermonecrosis, neutrophil infiltration, and endogenous gelatinase expression.J Invest Dermatol. 2005; 124: 725-731Google Scholar). Therefore, further in vivo investigations focused on the control of IL-8 expression after envenomation of spider venom or SMD could be of critical to the understanding of the pathophysiology of loxoscelism and indentify novel mechanism for therapeutic intervention. The authors state no conflict of interest.

We report the cloning of sphingomyelinase D (SMD) cDNA from Loxosceles reclusa, Loxosceles boneti and Loxosceles laeta into bacterial expression systems, as well as optimization of expression conditions so as to obtain soluble and active recombinant enzymes. The recombinant mature SMDs, tagged with a histidine tail at the N- or C-termini, were compared in terms of toxicity and enzymatic activity, and were used as immunogens for the production of monovalent antisera in rabbits and F(ab′)2 preparations in animals used for commercial antivenom production (horses). We performed studies on in vitro inhibition of enzymatic activity of natural venom preparations by antibodies generated against the tagged proteins. We also present and discuss the results of studies on the specific and para-specific in vivo protective potential of the rabbit and equine antibody preparations against the recombinant proteins themselves and natural venom preparations. Our conclusions support the feasibility of using recombinant SMDs for production and evaluation of polyvalent anti-Loxosceles antivenoms, and we offer data on the potential of paraspecific neutralization in the context of the antigenic groupings and the molecular phylogeny of those active SMDs for which amino acid sequence information is available.

Peptide nucleic acids (PNAs) may be a potent tool for gene function studies in medically important parasitic organisms, especially those that have not before been accessible to molecular genetic knockout approaches. One such organism is Entamoeba histolytica, the causative agent of amebiasis, which infects about 500 million people and is the cause of clinical disease in over 40 million each year, mainly in the tropical and subtropical world. We used PNA antisense oligomers to inhibit expression of an episomally expressed gene (neomycin phosphorotransferase, NPT) and a chromosomal gene (EhErd2, a homolog of Erd2, a marker of the Golgi system in eukaryotic cells) in axenically cultured trophozoites of E. histolytica. Measurement of NPT enzyme activity and EhErd2 protein levels, as well as measurement of cellular proliferation, revealed specific decreases in expression of the target genes, and concomitant inhibition of cell growth, in trophozoites treated with micromolar concentrations of unmodified antisense PNA oligomers.