Description of Loxtox protein family and identification of a new group of Phospholipases D from Loxosceles similis venom gland.

Envenoming resulting from Loxosceles spider bites (loxoscelism) is a recognized public health problem in Brazil. However, the pathophysiology of loxoscelism caused by L. similis bites, which is widespread in Brazil, remains poorly understood. In the present work, the RNA sequencing (RNA-Seq - Next Generation sequencing - NGS) of the L. similis venom gland was performed to identify and analyze the sequences of the key component phospholipase D. The sequences were aligned based on their classical domains, and a phylogenetic tree was constructed. In the bioinformatics analysis, 23 complete sequences of phospholipase D proteins were found and classified as Loxtox proteins, as they contained the characteristic domains of phospholipase D: the active site, the Mg(2+)-binding domain, and the catalytic loop. Three phospholipase D sequences with non-canonical domains were also found in this work. They were analyzed separately and named PLDs from L. similis (PLD-Ls). This study is the first to characterize phospholipase D sequences from Loxosceles spiders by RNA-Seq. These results contribute new knowledge about the composition of L. similis venom, revealing novel tools that could be used for pharmacological, immunological, and biotechnological applications.

Evolution of alternative methodologies of scorpion antivenoms production.

Scorpionism represents a serious public health problem resulting in the death of children and debilitated individuals. Scorpion sting treatment employs various strategies including the use of specific medicines such as antiserum, especially for patients with severe symptoms. In 1909 Charles Todd described the production of an antiserum against the venom of the scorpion Buthus quinquestriatus. Based on Todd's work, researchers worldwide began producing antiserum using the same approach i.e., immunization of horses with crude venom as antigen. Despite achieving satisfactory results using this approach, researchers in this field have developed alternative approaches for the production of scorpion antivenom serum. In this review, we describe the work published by experts in toxinology to the development of scorpion venom antiserum. Methods and results describing the use of specific antigens, detoxified venom or toxins, purified toxins and or venom fractions, native toxoids, recombinant toxins, synthetic peptides, monoclonal and recombinant antibodies, and alternative animal models are presented.

Comparison of silver nitrate and tetracycline as pleural sclerosing agents in rabbits.

The ideal agent to produce pleurodesis has not been identified. Tetracycline, the drug used most commonly in the 1980s, is no longer available. Talc either aerosolized or in a slurry is the agent used just most commonly at the present time, but there are concerns about its safety. Another possibility is silver nitrate, which was widely used in the past, but was abandoned on account of side effects. We hypothesized that lower concentrations of silver nitrate than had been used in the past would be effective in creating a pleurodesis in rabbits. The following medications in a total volume of 2 mL were instilled intrapleurally in three groups of ten anesthetized rabbits: 0.25% or 0.50% silver nitrate and 35 mg/kg tetracycline. Twenty-eight days after the injection, the animals were sacrificed and the pleural spaces were assessed grossly for evidence of pleurodesis and microscopically for evidence of fibrosis and inflammation. The intrapleural injection of 0.50% silver nitrate produced an effective pleurodesis. The mean degree of gross pleurodesis in the rabbits that received 0.50% silver nitrate (3.4 +/- 1.2) did not differ significantly from that of the rabbits that received tetracycline (3.5 +/- 0.7) (scale 0 to 4). The mean degree of microscopic pleural fibrosis in the rabbits that received 0.50% silver nitrate (3.4 +/- 0.7) did not differ significantly from that of the rabbits that received tetracycline (3.9 +/- 0.3). However, 0.25% silver nitrate was ineffective in creating pleural fibrosis, either grossly or microscopically. No rabbits died after the intrapleural injection of the drugs. There were no observed side effects after the injection of silver nitrate. The present study demonstrates that 0.50% silver nitrate instilled into the pleural space is an effective agent for producing pleurodesis in the rabbit; its effect is comparable to tetracycline 35 mg/kg. This agent should be compared with tetracycline derivatives and talc in studies in humans.

Relationship between pleural fluid and serum cholesterol levels.

INTRODUCTION: Since the criteria of Light and colleagues for differentiating transudates and exudates were described, other tests, including the pleural fluid (PF) cholesterol test, have been proposed for the same purpose. However, the factors influencing PF cholesterol levels have not been clearly delineated. PURPOSE: To analyze the relationships among total cholesterol (CHOL), low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides (TRIG) in serum (S) and PF. METHODS: PF and S from 99 patients (transudates, 13 patients; exudates, 86 patients) were analyzed for CHOL, HDL, LDL, TRIG, apolipoprotein AI, apolipoprotein B, and protein. The relationship between the PF and S level for each of these measurements was analyzed with linear regression and multiple regression using the ratio of PF to S protein for that measurement as a second independent variable. RESULTS: This study demonstrated that CHOL levels in PF are related to S cholesterol levels and to the permeability of the pleura (r = 0.88; p < 0.001). However, the percentage of CHOL associated with LDL and HDL (56%) in the PF was much lower than that associated with LDL and HDL in S (93%), suggesting that lipoproteins are modified once they enter the pleural space. The PF TRIG was not closely related to its S level or to the PF/S protein ratio (r = 0.49). CONCLUSION: PF cholesterol levels can be closely predicted from the S cholesterol levels and the permeability of the pleura, as reflected by the ratio of PF protein to S protein. Therefore, the CHOL ratio should not provide additional information to that provided by the protein ratio when trying to differentiate transudates from exudates. PF lipoproteins (LDL and HDL) undergo metabolic alterations once they enter the pleural space. PF TRIG levels are not closely related to S levels or to the permeability of the pleura.

Silver nitrate is superior to talc slurry in producing pleurodesis in rabbits.

STUDY OBJECTIVE: The ideal agent for producing pleurodesis has not been identified. Although talc is the agent most commonly used at the present time, there are concerns about its safety. Silver nitrate is a possible alternative agent. The purpose of the present study was to compare the effectiveness of intrapleural silver nitrate and talc slurry in producing pleurodesis in rabbits. Additionally, the total amount of pleural collagen and the distribution of thick and thin collagen fibers were studied. DESIGN: Two groups of 10 rabbits received either 0.50% silver nitrate or 400 mg/kg talc in a total volume of 2 mL intrapleurally. The animals were killed 28 days after injection, and the pleural spaces were assessed grossly for evidence of pleurodesis and microscopically for evidence of inflammation and fibrosis. Collagen was assessed with the van Gieson's and picrosirius stains. RESULTS: The macroscopic pleurodesis (scale, 0 to 4; mean +/- SEM) resulting from the intrapleural injection of silver nitrate (3.4+/-0.2) was significantly better (p<0.001) than that resulting from talc (1.6+/- 0.1). The mean degree of microscopic pleural fibrosis induced by silver nitrate (3.3+/-0.3) was significantly higher (p = 0.003) than that induced by talc (1.8+/-0.1). The mean amount of microscopic pleural collagen (van Gieson's) was significantly greater (p<0.001) in the rabbits that received silver nitrate (3.0+/-0.2) than in those that received talc (1.6+/-0.2). The distribution of thick and thin collagen fibers did not differ between the groups. CONCLUSIONS: We conclude that, in our rabbit model, intrapleural silver nitrate was more effective than talc in producing a pleurodesis.

Experimental pleurodesis in rabbits induced by silver nitrate or talc: 1-year follow-up.

STUDY OBJECTIVE: To compare the pleurodesis results from the intrapleural injection of silver nitrate and talc slurry over an observation period of 12 months in rabbits. DESIGN: Rabbits were randomized to receive 2 mL of 0.5% silver nitrate or 400 mg/kg of talc slurry in 2 mL intrapleurally. Ten rabbits in each group were killed at 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, and 12 months after intrapleural injection. The degree of gross pleurodesis and the amount of microscopic pleural fibrosis and inflammation were graded on a scale of 0 to 4. RESULTS: The mean +/- SEM gross pleurodesis score in the 70 rabbits that received silver nitrate was 3.34 +/- 0.08, which was significantly higher than the score of 2.32 +/- 0.09 in the 70 rabbits that received talc. The mean gross pleurodesis score was significantly higher at each of the observation times (p < 0.05), except at 2 months, in the rabbits that received silver nitrate. The pleurodesis was distributed throughout the thorax in the rabbits that received silver nitrate, while it was only in the ventral thorax in the rabbits that received talc slurry. The gross pleurodesis scores showed no tendency to decrease during the 12-month observation period in either treatment group. The persistence of talc in the pleural space did not lead to chronic inflammatory changes because the inflammation scores were similar in both groups at all observation times. The microscopic pleural fibrosis score tended to decrease with time in the silver nitrate group but not in the talc slurry group. CONCLUSIONS: The intrapleural injection of 2 mL of 0.5% silver nitrate produces a better pleurodesis than does the intrapleural injection of 400 mg/kg of talc slurry in rabbits. The pleurodesis induced by silver nitrate persists for at least 1 year. The efficacy of silver nitrate as a sclerosing agent in humans should be evaluated.

Molecular and functional characterization of metalloserrulases, new metalloproteases from the Tityus serrulatus venom gland.

Tityus serrulatus is a Brazilian scorpion species with great medical significance. While the effects of neurotoxins have been extensively studied, little is known about the proteases expressed in the venom gland of this arthropod. In this study, clones from a T. serrulatus (Ts) venom gland cDNA library were selected according to homology to proteases. The sequences were aligned in the database and classified by homology. Similarity and identity analyses of the sequences were carried out, and a phylogenetic tree was constructed with the sequences of other proteases. These cDNA sequences correspond to ten different metalloproteases, named metalloserrulases (TsMS). TsMS 1-9 belong to the metzincin family, which has three domains: signal peptide, propeptide, and metalloprotease domain; while TsMS 10 belongs to the gluzincin family. The proteolytic activity of the venom was inferred from the cleavage of fibrinogen, and the residues recognized by the proteases were determined by cleavage of a tripeptide library using a fluorescence resonance energy transfer assay. The Ts venom showed proteolytic activity on fibrinogen and preferential cleavage close to the basic residues K and R. Its activity could be inhibited by EDTA, indicating that the venom from this scorpion predominantly consists of metalloproteases.

ADP is a vasodilator component from Lasiodora sp. mygalomorph spider venom.

Members of the spider genus Lasiodora are widely distributed in Brazil, where they are commonly known as caranguejeiras. Lasiodora spider venom is slightly harmful to humans. The bite of this spider causes local pain, edema and erythema. However, Lasiodora sp. spider venom may be a source of important pharmacological tools. Our research group has described previously that Lasiodora sp. venom produces bradycardia in the isolated rat heart. In the present work, we sought to evaluate the vascular effect of Lasiodora sp. venom and to isolate the vasoactive compounds from the venom. The results showed that Lasiodora spider venom induced a concentration-dependent vasodilation in rat aortic rings, which was dependent on the presence of a functional endothelium and abolished by the nitric oxide synthase (NOS) inhibitor L-NAME. Western blot experiments revealed that the venom also increased endothelial NOS function by increasing phosphorylation of the Ser¹¹77 residue. Assay-directed fractionation isolated a vasoactive fraction from Lasiodora sp. venom. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) assays identified a mixture of two compounds: adenosine diphosphate (ADP, approximately 90%) and adenosine monophosphate (AMP, approximately 10%). The vasodilator effects of Lasiodora sp. whole venom, as well as ADP, were significantly inhibited by suramin, which is a purinergic P2-receptor antagonist. Therefore, the results of the present work indicate that ADP is a main vasodilator component of Lasiodora sp. spider venom.

Evaluation of Brazilian biotechnology patent activity from 1975 to 2010.

The analysis of patent activity is one methodology used for technological monitoring. In this paper, the activity of biotechnology-related patents in Brazil were analyzed through 30 International Patent Classification (IPC) codes published by the Organization for Economic Cooperation and Development (OECD). We developed a program to analyse the dynamics of the major patent applicants, countries and IPC codes extracted from the Brazilian Patent Office (INPI) database. We also identified Brazilian patent applicants who tried to expand protection abroad via the Patent Cooperation Treaty (PCT). We had access to all patents published online at the INPI from 1975 to July 2010, including 9,791 biotechnology patent applications in Brazil, and 163 PCTs published online at World Intellectual Property Organization (WIPO) from 1997 to December 2010. To our knowledge, there are no other online reports of biotechnology patents previous to the years analyzed here. Most of the biotechnology patents filed in the INPI (10.9%) concerned measuring or testing processes involving nucleic acids. The second and third places belonged to patents involving agro-technologies (recombinant DNA technology for plant cells and new flowering plants, i.e. angiosperms, or processes for obtaining them, and reproduction of flowering plants by tissue culture techniques). The majority of patents (87.2%) were filed by nonresidents, with USA being responsible for 51.7% of all biotechnology patents deposited in Brazil. Analyzing the resident applicants per region, we found a hub in the southeast region of Brazil. Among the resident applicants for biotechnology patents filed in the INPI, 43.5% were from São Paulo, 18.3% were from Rio de Janeiro, and 9.7% were from Minas Gerais. Pfizer, Novartis, and Sanofi were the largest applicants in Brazil, with 339, 288, and 245 biotechnology patents filed, respectively. For residents, the largest applicant was the governmental institution FIOCRUZ (Oswaldo Cruz Foundation), which filed 69 biotechnology patents within the period analyzed. The first biotechnology patent applications via PCT were submitted by Brazilians in 1997, with 3 from UFMG (university), 2 from individuals, and 1 from EMBRAPA (research institute).

A new look at old agents for pleurodesis: nitrogen mustard, sodium hydroxide, and silver nitrate.

In this review we analyze the evolution of pleurodesis. In spite of the fact that this procedure started in the beginning of the 20th century, the ideal sclerosing agent is not yet known. Emphasis is placed on the current tendency toward minimally invasive procedures in which insertion of catheters is favored over surgical procedures such as placement of chest tubes or thoracoscopy. Among the sclerosing agents, talc is preferred throughout the world. However, the possible development of acute respiratory distress syndrome, which is sometimes fatal, caused the awakening of interest in other drugs. Nitrogen mustard induces pleurodesis but causes important side effects. Sodium hydroxide and silver nitrate are effective and may be used in humans beings.

Corynebacterium parvum versus tetracycline as pleural sclerosing agents in rabbits.

Tetracycline has been one of the most commonly used agents for producing a pleurodesis. However, it is no longer available due to more stringent requirements on the manufacturing process. The objective of this project was to determine whether Corynebacterium parvum is an effective sclerosant in an experimental model in rabbits. The following medications were instilled intrapleurally in anaesthetized male rabbits: tetracycline 35 mg.kg-1 or C. parvum 4 or 8 mg, all diluted with bacteriostatic saline solution. Twenty eight days after the instillation, the animals were sacrificed and the pleural spaces assessed macroscopically for evidence of pleurodesis and microscopically for evidence of fibrosis and inflammation. The intrapleural injection of C. parvum was ineffective in creating pleural fibrosis. The mean degree of pleurodesis in the 10 rabbits who received tetracycline was 3.5 +/- 0.7 (scale 0-4) whilst in the 10 rabbits that received 4 mg C. parvum it was 0.0 +/- 0.0, and in the 10 rabbits that received 8 mg C. parvum it was 0.5 +/- 0.8. Based on this study, we recommend that C. parvum should not be used as a pleural sclerosant in patients with normal pleura.

Effectiveness of sodium hydroxide as a pleural sclerosing agent in rabbits: influence of concomitant intrapleural lidocaine.

The two agents that have been used most commonly to produce a pleurodesis are tetracycline and bleomycin. Tetracycline is no longer generally available because of more stringent requirements on the manufacturing process. Bleomycin is very expensive. Therefore, alternative agents are necessary particularly in developing countries. The objective of this project was to determine whether 0.5% sodium hydroxide is an effective sclerosant in an experimental model in rabbits. Sodium hydroxide (NaOH) (2 ml of 0.5%) was instilled intrapleurally in 24 anesthetized male rabbits. Half the rabbits received 1 ml of 2% lidocaine 3-5 min before the NaOH. Twenty-eight days after the instillation, the animals were sacrificed, and the pleural spaces were assessed grossly for evidence of pleurodesis and microscopically for evidence of fibrosis and inflammation. The results indicated that the intrapleural injection of NaOH was effective in creating a pleurodesis only if the animals were not premedicated with lidocaine. The mean (+/- S.D.) degree of gross pleurodesis after NaOH alone 2.8 (1.0) was significantly (p < 0.001) greater than after that following the combination 1.3 (0.5). We conclude that NaOH is an effective pleural sclerosant but that it is ineffective if it is injected concomitantly with lidocaine.

Effectiveness of ethanolamine oleate as a pleural sclerosing agent in rabbits.

The ideal pleural sclerosing agent should be easily administered, without significant side effects, inexpensive, and widely available. None of the agents presently used meets all of these criteria. Ethanolamine oleate (ETH) is a sclerosing agent used in the sclerotherapy treatment of varicose veins of the legs and esophagus. The objective of the present study was to assess the efficacy of ETH as a pleural sclerosant in rabbits. An additional objective was to assess if better results were obtained when dextrose 50% (D50) as opposed to saline was used as the diluent. Each group of 10 rabbits received a total volume of 2 ml intrapleurally. The eight treatments were as follows: (1) 2 ml saline; (2) 2 ml D50; (3) 25 mg ETH plus 1.5 ml saline; (4) 25 mg ETH plus 1.5 ml D50; (5) 50 mg ETH plus 1.0 ml saline; (6) 50 mg ETH plus 1 ml D50; (7) 75 mg ETH plus 0.5 ml D50, and (8) 100 mg ETH. The rabbits were sacrificed 28 days after the injection. The intrapleural instillation of ETH resulted in evident pleurodesis, which was dose-dependent; 100 mg ETH induced significantly (p<0.05) more adhesions than did any other treatment. The selection of the diluent had no effect on the pleurodesis. The microscopic examination of the right visceral pleura showed that the mean degree of fibrosis after 100 mg ETH was significantly (p<0.05) greater than that after the other solutions. The mean degree of pleural inflammation, lung inflammation and lung fibrosis was minimal in all the groups. From this study we conclude that undiluted ETH produces pleurodesis in our experimental model. At the doses used, the pleurodesis was less than that produced after talc, tetracycline derivatives or silver nitrate in the same model.

Comparison of nitrogen mustard, cytarabine and dacarbazine as pleural sclerosing agents in rabbits.

We have previously shown that the intrapleural injection of mitozantrone but not bleomycin resulted in pleural fibrosis. Mechlorethamine hydrochloride (nitrogen mustard) was used extensively in the past to control malignant effusions, with relatively good success. The objective of this study was to determine if the intrapleural injection of nitrogen mustard would produce pleural sclerosis in our experimental model in rabbits. We therefore evaluated sclerosing capabilities of nitrogen mustard as well as those of cytarabine and dacarbazine. Nitrogen mustard (0.4 and 0.8 mg x kg(-1)), cytarabine (3, 6 and 20 mg x kg(-1)) and dacarbazine (4, 8 and 20 mg x kg(-1)) were instilled intrapleurally into anaesthetized rabbits. Twenty eight days after the instillation, the animals were killed, and the pleural spaces were assessed grossly for evidence of pleurodesis and microscopically for evidence of fibrosis and inflammation. The intrapleural injection of 0.8 mg x kg(-1) nitrogen mustard was effective in creating pleural fibrosis, either grossly or microscopically. The mean degree (scale 0-4) of gross pleurodesis in the rabbits that received 0.8 mg x kg(-1) nitrogen mustard was 3.2+/-1.0 and the mean degree of microscopic pleural fibrosis was 3.5+/-0.8. The intrapleural injection of 0.4 mg x kg(-1) nitrogen mustard and the different doses of cytarabine (3, 6 and 20 mg x kg(-1)) and dacarbazine (4, 8 and 20 mg x kg(-1)) were ineffective in producing pleurodesis. From this study, we conclude that the intrapleural injection of 0.8 mg x kg(-1) of nitrogen mustard produces clinically significant pleurodesis in rabbits. Consideration should be given to future clinical studies utilizing 0.6-0.8 mg x kg(-1) nitrogen mustard intrapleurally for the treatment of malignant pleural effusion.

Effectiveness of silver nitrate compared to talc slurry as pleural sclerosing agent in rabbits. Influence of concomitant intrapleural lidocaine.

UNLABELLED: The ideal agent for producing pleurodesis has not been identified. Talc, the most commonly used, poses several problems. Another possibility is silver nitrate, which was widely used in the past. PURPOSE: To determine the influence of the intrapleural instillation of lidocaine in producing a pleurodesis with silver nitrate, to define the effect of lidocaine in the maturation of the collagen fibers, and to confirm that the pleurodesis after silver nitrate is stronger than after talc. METHODS: We studied three groups of 8 rabbits. Two groups received 0.5% silver nitrate; in one we had previously injected 0.5 ml of 2% lidocaine. The third group received 400 mg/kg talc (2 ml). The animals were sacrificed 28 days after the injection, and the pleural spaces were assessed grossly for evidence of pleurodesis and microscopically for evidence of inflammation and fibrosis. The total amount of pleural collagen and the distribution of thick and thin collagen fibers were quantified. Collagen was identified using picrosirius red stain. RESULTS: In the two groups that received silver nitrate (without lidocaine: 3.5 + 03 and with lidocaine: 3.2 + 0.3), the macroscopic pleurodesis (scale 0 - 4) was significantly (p = 0.001) better than that resulting from talc (1.6 + 0.2). The mean degree of pleural fibrosis induced by silver nitrate (3.5 + 0.2) was significantly (p = 0.004) higher than that induced by talc (1.9 + 0.1). The previous instillation of lidocaine resulted in a tendency for decreased amounts of fibrosis (3.1 + 0.4). The mean amount (10(3)mm2) of pleural collagen was significantly (p = 0.009) greater in the rabbits that received silver nitrate (116.9 + 22.7) than in those that received talc (10.7 + 3.4). The injection of lidocaine slightly reduced the collagen (80.1 + 30.3). The distribution of collagen fibers did not differ among the groups. CONCLUSION: This rabbit model clearly confirms that intrapleural silver nitrate is more effective than talc for producing pleurodesis. The previous intrapleural instillation of lidocaine results in a decreasing trend in the amount of collagen, but does not change the effectiveness of the pleural fusion or modify the process of collagen maturation.