RESUMO

Diabetic neuropathic pain is one of the complications that affect a wide variety of the diabetic population and is often difficult to treat. Only a small number of patients experience pain relief, which usually comes with onerous side effects and low levels of satisfaction. The search for new analgesic drugs is necessary, given the limitations that current drugs present. Combining drugs to treat neuropathic pain has been attracting interest to improve their efficacy compared to single-drug monotherapies while also reducing dose sizes to minimize side effects. The aim of our study was to verify the antinociceptive effect of a synthetic peptide, PnPP-15, alone and combined with pregabalin, in male Swiss diabetic mice using the von Frey method. PnPP-15 is a synthetic peptide derived from PnPP19, a peptide representing a discontinuous epitope of the primary structure of the toxin PnTx2-6 from the venom of the spider Phoneutria nigriventer. The antinociceptive activity of both compounds was dose-dependent and showed synergism, which was verified by isobolographic analysis. Treatment with PnPP-15 did not cause spontaneous or forced motor changes and did not cause any damage or signs of toxicity in the analyzed organs (pancreas, lung, heart, kidney, brain, or liver). In conclusion, PnPP-15 is a great candidate for an analgesic drug against neuropathic pain caused by diabetes and exerts a synergistic effect when combined with pregabalin, allowing for even more efficient treatment.

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RESUMO

Dendritic cells (DCs) vaccine is a potential tool for oncoimmunotherapy. However, it is known that this therapeutic strategy has failed in solid tumors, making the development of immunoadjuvants highly relevant. Recently, we demonstrated that Phoneutria nigriventer spider venom (PnV) components are cytotoxic to glioblastoma (GB) and activate macrophages for an antitumor profile. However, the effects of these molecules on the adaptive immune response have not yet been evaluated. This work aimed to test PnV and its purified fractions in DCs in vitro. For this purpose, bone marrow precursors were collected from male C57BL6 mice, differentiated into DCs and treated with venom or PnV-isolated fractions (F1-molecules < 3 kDa, F2-3 to 10 kDa and F3->10 kDa), with or without costimulation with human GB lysate. The results showed that mainly F1 was able to activate DCs, increasing the activation-dependent surface marker (CD86) and cytokine release (IL-1ß, TNF-α), in addition to inducing a typical morphology of mature DCs. From the F1 purification, a molecule named LW9 was the most effective, and mass spectrometry showed it to be a peptide. The present findings suggest that this molecule could be an immunoadjuvant with possible application in DC vaccines for the treatment of GB.

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RESUMO

The phylum Arthropoda comprises approximately 85% of all described animal species. The class Arachnida includes some invertebrates of great importance as they are either involved in the transmission of diseases or poses a risk of human envenomation. Spiders belonging to the genus Phoneutria sp., are the arachnids exhibiting medical importance. These animals were quarantined and maintained in captivity at the Biotério de Artrópodes of the Instituto Butantan, São Paulo, Brazil, for the production of the anti-arachnid serum. A total 509 feces samples from different Phoneutria nigriventer were analyzed, and 131 (25.73%) samples were found to be positive for flagellates and ciliates. All positive samples were subjected to DNA extraction and amplification of 18S gene. A total of 16 sequences were obtained and analyzed using BLAST. Sequences were identified as Colpoda steiini, one as Colpoda aspera, one to Colpoda sp., and one as "ciliated". Four identified as Parabodo caudatus, two as Urostipulosphaera sp., one as Helkesimastix sp., and one as a Euglena-like. The presence of clinical signs was observed in 16 spiders. The intestinal protozoa that affect armed spiders were identified for the first time as an initial step for understanding the parasitic diseases in these organisms.

RESUMO

The species Phoneutria nigriventer belongs to the class Arachnida and order aranae, it is described as a synanthropic animal, with characteristics of aggressiveness, high stress, wanderers and opportunists. In addition, it has a medical interest in public health, having a potential poison to cause injuries, not only in predatory prey, but also in humans. The developmental stages of the species are, in sequence: ootheca, egg, pre-larval, larval, young, juvenile, pre-adult and adult. In the present study, the phases of ootheca and, mainly, hatchlings were analyzed, through a creation in captivity with constant control of physical parameters (temperature and humidity), without significant variations. Thus, through follow-up, it is possible to understand aspects that influence development, to ensure the success of individuals until sexual maturation. As a sample number we obtained an N=50, among these, some arranged in the arthropod vivarium via the external environment and a large majority built internally by pregnant females. To effect the hatching of the oothecae, they were allocated in a nursery, with substrates and essential materials that mimic a natural, dark and humidified environment. Thus, 40 oothecae were designated internally, being thirty-three viable, six not viable and one partially viable, the remaining 10 came from the external environment with seven viable and three not viable. In general, the number of individualized individuals ranged from 9 to 1652, with hatching times ranging from 17 to 74 days and dates from hatching to the effective individualizing action from 14 to 45 days. The dimensioning of the volume and weight of the eggs was also defined through previously established formulas. Therefore, when comparing with other results, it was noticed that the number of individuals exceeded expectations and the hatching date fit the observed, with only one outline in the research sample.

A espécie Phoneutria nigriventer enquadra-se na classe Arachnida e ordem aranae, ela é descrita como um animal sinantrópico, com características de agressividade, elevado estresse, errantes e oportunistas. Além disso, possui interesse médico na saúde pública, tendo um veneno potencial para causar injúrias, não apenas em presas-predadores, como também nos seres humanos. Os estágios de desenvolvimento da espécie são, em sequência: ooteca, ovo, pré-larval, larval, filhotes, jovens, pré- adulto e adulto. No presente estudo, foram analisados as fases de ootecas e, principalmente, filhotes, através de uma criação em cativeiro com controle constante de parâmetros físicos (temperatura e umidade), sem variações significativas. Assim, pelo acompanhamento pode-se compreender aspectos que influenciam o desenvolvimento, para garantir o sucesso dos indivíduos até maturação sexual. Como número amostral obtivemos um N=50, dentre essas, algumas dispostas no biotério de artrópodes via meio externo e, uma grande maioria construídas internamente por fêmeas prenhas. Para efetivação da eclosão das ootecas, estas foram alocadas em um viveiro, com substratos e materiais essenciais que mimetizem um ambiente natural, escuro e umidificado. Dessa forma, 40 ootecas foram designadas internamente, sendo trinta e três viáveis, seis inviáveis e uma parcialmente viável, as 10 restantes vieram de meio externo com sete viáveis e três inviáveis. De modo geral, o número de indivíduos individualizados variou de 9 a 1652, com tempos de eclosão no intervalo de 17 a 74 dias e datas desde a eclosão até a efetiva ação de individualizar de 14 a 45 dias. Definiu-se, também, o dimensionamento de volume e peso dos ovos através de fórmulas estabelecidas previamente. Portanto, ao comparar com outros resultados, percebeu-se que o número de indivíduos superou as expectativas e, a data de eclosão encaixou-se no observado, com apenas um outline na amostragem da pesquisa.

RESUMO

The phylum Arthropoda comprises approximately 85% of all described animal species. The class Arachnida includes some invertebrates of great importance as they are either involved in the transmission of diseases or poses a risk of human envenomation. Spiders belonging to the genus Phoneutria sp., are the arachnids exhibiting medical importance. These animals were quarantined and maintained in captivity at the Biotério de Artrópodes of the Instituto Butantan, São Paulo, Brazil, for the production of the anti-arachnid serum. A total 509 feces samples from different Phoneutria nigriventer were analyzed, and 131 (25.73%) samples were found to be positive for flagellates and ciliates. All positive samples were subjected to DNA extraction and amplification of 18S gene. A total of 16 sequences were obtained and analyzed using BLAST. Sequences were identified as Colpoda steiini, one as Colpoda aspera, one to Colpoda sp., and one as “ciliated”. Four identified as Parabodo caudatus, two as Urostipulosphaera sp., one as Helkesimastix sp., and one as a Euglena-like. The presence of clinical signs was observed in 16 spiders. The intestinal protozoa that affect armed spiders were identified for the first time as an initial step for understanding the parasitic diseases in these organisms.

RESUMO

Immunomodulation has been considered an important approach in the treatment of malignant tumours. However, the modulation of innate immune cells remains an underexplored tool. Studies from our group demonstrated that the Phoneutria nigriventer spider venom (PnV) administration increased the infiltration of macrophage in glioblastoma, in addition to decreasing the tumour size in a preclinical model. The hypothesis that PnV would be modulating the innate immune system led us to the main objective of the present study: to elucidate the effects of PnV and its purified fractions on cultured macrophages. Results showed that PnV and the three fractions activated macrophages differentiated from bone marrow precursors. Further purification generated 23 subfractions named low weight (LW-1 to LW-12) and high weight (HW-1 to HW-11). LW-9 presented the best immunomodulatory effect. Treated cells were more phagocytic, migrated more, showed an activated morphological profile and induced an increased cytotoxic effect of macrophages on tumour cells. However, while M1-controls (LPS) increased IL-10, TNF-alpha and IL-6 release, PnV, fractions and subfractions did not alter any cytokine, with the exception of LW-9 that stimulated IL-10 production. These findings suggest that molecules present in LW-9 have the potential to be used as immunoadjuvants in the treatment of cancer.

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Fibromyalgia is characterized by the amplification of central nervous system pain with concomitant fatigue, sleep, mood disorders, depression, and anxiety. It needs extensive pharmacological therapy. In the present study, Swiss mice were treated with reserpine (0.25 mg/kg, s.c.) over three consecutive days, in order to reproduce the pathogenic process of fibromyalgia. On day 4, the administrations of the Tx3-3 toxin produced significant antinociception in the mechanical allodynia (87.16% ±12.7%) and thermal hyperalgesia (49.46% ± 10.6%) tests when compared with the PBS group. The effects produced by the classical analgesics (duloxetine 30 mg/kg, pramipexole 1 mg/kg, and pregabalin 30 mg/kg, p.o., respectively) in both of the tests also demonstrated antinociception. The administrations were able to increase the levels of the biogenic amines (5-HTP and DE) in the brain. The treatments with pramipexole and pregabalin, but not duloxetine, decreased the immobility time in the FM-induced animals that were submitted to the forced swimming test; however, the Tx3-3 toxin (87.45% ± 4.3%) showed better results. Taken together, the data has provided novel evidence of the ability of the Tx3-3 toxin to reduce painful and depressive symptoms, indicating that it may have significant potential in the treatment of FM.

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BACKGROUND: Glioblastoma (GB) cells have the ability to migrate and infiltrate the normal parenchyma, leading to the formation of recurrent tumors often adjacent to the surgical extraction site. We recently showed that Phoneutria nigriventer spider venom (PnV) has anticancer effects mainly on the migration of human GB cell lines (NG97 and U-251). The present work aimed to investigate the effects of isolated components from the venom on migration, invasiveness, morphology and adhesion of GB cells, also evaluating RhoA-ROCK signaling and Na+/K+-ATPase ß2 (AMOG) involvement. METHODS: Human (NG97) GB cells were treated with twelve subfractions (SFs-obtained by HPLC from PnV). Migration and invasion were evaluated by scratch wound healing and transwell assays, respectively. Cell morphology and actin cytoskeleton were shown by GFAP and phalloidin labeling. The assay with fibronectin coated well plate was made to evaluate cell adhesion. Western blotting demonstrated ROCK and AMOG levels and a ROCK inhibitor was used to verify the involvement of this pathway. Values were analyzed by the GraphPad Prism software package and the level of significance was determinate using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test. RESULTS: Two (SF1 and SF11) of twelve SFs, decreased migration and invasion compared to untreated control cells. Both SFs also altered actin cytoskeleton, changed cell morphology and reduced adhesion. SF1 and SF11 increased ROCK expression and the inhibition of this protein abolished the effects of both subfractions on migration, morphology and adhesion (but not on invasion). SF11 also increased Na+/K+-ATPase ß2. CONCLUSION: All components of the venom were evaluated and two SFs were able to impair human glioblastoma cells. The RhoA effector, ROCK, was shown to be involved in the mechanisms of both PnV components. It is possible that AMOG mediates the effect of SF11 on the invasion. Further investigations to isolate and biochemically characterize the molecules are underway.

RESUMO

BACKGROUND: PnPa11 and PnPa13 are synthetic peptides derived from Phoneutria nigriventer spider venom, which display antinociceptive and neuroprotective properties. In this work, we evaluated the safety of intravitreal use and the neuroprotective effect of these peptides. METHODS: The cytotoxicity and the antiangiogenic activity of these peptides were evaluated by the sulforhodamine-B method and chicken chorioallantoic membrane (CAM) assay, respectively. The in vivo safety was analyzed in Wistar rats that were intravitreally injected with different doses (0.50; 1.25; 2.50; 3.75 and 5.00 µg/mL) of these peptides (right eye, n = 6). The retinal function was assessed by electroretinography exams (ERG), intraocular pressure (IOP), and histological analyzes. In order to investigate the neuroprotective effect, Wistar rats received intravitreal injections (right eye, n = 6) of peptides at 1.25 µg/mL and then were exposed to blue LED light. In addition, the visual function and the retinal microstructure were verified. RESULTS: Cytotoxicity analyses demonstrated that the peptides did not present any toxicity over ARPE-19 (adult retinal pigmented epithelial) cell line and the antiangiogenic study highlighted that the peptides promoted the reduction of blood vessels. The intravitreal injection did not cause major changes, neither induced any irreversible damage. In the retinal degeneration assay, the ERG records demonstrated that the prior treatment with PnPa11 and PnPa13 protected the retina from damage. Morphological analyses confirmed the ERG findings. Immunoblotting analyses revealed that PnPa11 increased Erk1/2, NR2A, and NR2B retinal expression after the light stress model, but did not cause Akt1 activation, while PnPa13 prevented Erk1/2 and Akt1 dephosphorylation. CONCLUSIONS: The intraocular administration of these peptides was well tolerated and presented protective activity against retinal degeneration, suggesting the potential use of these peptides as neuroprotectors in the ophthalmological field.

RESUMO

BACKGROUND: Orofacial pain is clinically challenging, having therapeutic failures and side effects. This study evaluated the antinociceptive activities of the CTK 01512-2 toxin, the TRPA1 channel antagonist, and the selective inhibitor of the N-type voltage-gated calcium channels (N-type VGCC), in different pain models. MATERIALS AND METHODS: The trigeminal ganglia were stimulated in vitro with capsaicin. The in vivo models received subcutaneous (sc) injections of formalin into the upper lip of the rats, Freund's Complete Adjuvant (FCA) into the temporomandibular joint (TMJ), and infraorbital nerve constrictions (IONC). CTK 01512-2 at concentrations of 30, 100, and 300 pmol/site, intrathecally (ith), and MVIIA at 10, 30, and 100 pmol/site in the formalin test, guided the doses for the models. The glutamate levels in the CSF of the rats that were submitted to IONC were analyzed. RESULTS: CTK 01512-2 decreased the nociceptive behavior in the inflammatory phase of the formalin test (65.94 ± 7.35%) and MVIIA in the neurogenic phase (81.23 ± 3.36%). CTK 01512-2 reduced facial grooming with FCA in the TMJ (96.7 ± 1.6%), and in the IONC neuropathy model, it decreased heat hyperalgesia (100%) and cold hyperalgesia (81.61 ± 9.02%). The levels of glutamate in the trigeminal ganglia in vitro (81.40 ± 8.59%) and in the CSF in vivo (70.0 ± 9.2%) were reduced. CONCLUSIONS: The roles of TRPA1 in pain transduction and the performance of CTK 01512-2 in the inhibition of the N-type VGCCs were reinforced. This dual activity may represent an advantage in clinical treatments.

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