Protonectin (1-6): A novel chemotactic peptide from the venom of the social wasp Agelaia pallipes pallipes

Peptides constitute the largest group of Hymenoptera venom toxins; some of them interact with GPCR, being involved with the activation of different types of leukocytes, smooth muscle contraction and neurotoxicity. Most of these toxins vary from dodecapeptides to tetradecapeptides, amidated at their C-teminal amino acid residue. The venoms of social wasps can also contains some tetra-, penta-, hexa- and hepta-peptides, but just a few of them have been structurally and functionally characterized up to now. Protonectin (ILGTILGLLKGL-NH₂) is a polyfunctional peptide, presenting mast cell degranulation, release of lactate dehydrogenase (LDH) from mast cells, antibiosis against Gram-positive and Gram-negative bacteria and chemotaxis for polymorphonucleated leukocytes (PMNL), while Protonectin (1-6) (ILGTIL-NH₂) only presents chemotaxis for PMNL. However, the mixture of Protonectin (1-6) with Protonectin in the molar ratio of 1:1 seems to potentiate the biological activities dependent of the membrane perturbation caused by Protonectin, as observed in the increasing of the activities of mast cell degranulation, LDH releasing from mast cells, and antibiosis. Despite both peptides are able to induce PMNL chemotaxis, the mixture of them presents a reduced activity in comparison to the individual peptides. Apparently, when mixed both peptides seems to form a supra-molecular structure, which interact with the receptors responsible for PMNL chemotaxis, disturbing their individual docking with these receptors. In addition to this, a comparison of the sequences of both peptides suggests that the sequence ILGTIL is conserved, suggesting that it must constitute a linear motif for the structural recognition by the specific receptor which induces leukocytes migration.     

NMR methods for determining disulfide-bond connectivities

Animal toxins are the major class of secreted disulfide-rich proteins, with ∼70% containing two or more disulfide bonds. Incorrect pairing of these disulfide bonds typically leads to a non-native three-dimensional fold accompanied by a loss of protein function. Determination of the native disulfide-bond framework is therefore a key component in the structural characterization of toxins. In this article, we review NMR approaches for elucidation of disulfide-bond connectivities. A major advantage of these NMR approaches is that they are non-invasive, leaving the sample intact at the end of the analysis for use in other studies.     

Inhibition of Na currents by the toxin extracts from puffer fishes captured in the sea coast of Japan

The inhibitory effects of toxin extracted from muscle or liver of five different puffer fishes (hereafter referred as puffer(s)) captured on the Japanese sea coast were examined on voltage-dependent sodium current (I_Na) recorded from dissociated single rat hippocampal CA1 neurons. The inhibitory effects estimated from IC₅₀ values of toxin extracts on I_Na were in the order of Takifugu vermicularis > Lagocephalus wheeleri > Canthigaster rivulata > Takifugu rubripes > Arothron reticularis from muscle and T. vermicularis > T. rubripes > L. wheeleri > A. reticularis > C. rivulata from liver, thereby indicating that the amount of toxin in the liver or muscle differs between puffers. In addition, the present results indicate that the muscle of T. vermicularis, which is eaten in Japan, contains relatively higher amounts of toxin compared to those of T. rubripes, also eaten. This observation suggests that caution should be taken concerning the maximal edible amount of muscle prepared from T. rubripes.     

Pharmacological and biophysical properties of Ca2+ channels and subtype distributions in human adrenal chromaffin cells

In this study, we explored the pharmacological and biophysical properties of voltage-activated Ca(2+) channels in human chromaffin cells using the perforated-patch configuration of the patch-clamp technique. According to their pharmacological sensitivity to Ca(2+) channel blockers, cells could be sorted into two groups of similar size showing the predominance of either N- or P/Q-type Ca(2+) channels. R-type Ca(2+) channels, blocked by 77% with 20 muM Cd(2+) and not affected by 50 muM Ni(2+), were detected for the first time in human chromaffin cells. Immunocytochemical experiments revealed an even distribution of alpha (1E) Ca(2+) channels in these cells. With regard to their biophysical properties, L- and R-type channels were activated at membrane potentials that were 15-20 mV more negative than P/Q- and N-type channels. Activation time constants showed no variation with voltage for the L-type channels, decreased with increasing potentials for the R- and P/Q-type channels, and displayed a bell shape with a maximum at 0 mV for the N-type channels. R-type channels were also the most inactivated channels. We thus show here that human chromaffin cells possess all the Ca(2+) channel types described in neurons, L, N, P/Q, and R channels, but the relative contributions of N and P/Q channels differ among cells. Given that N- and P/Q-type Ca(2+) channel types can be differentially modulated, these findings suggest the possibility of cell-specific regulation in human chromaffin cells.     

Protective immunity to ricin toxin conferred by antibodies against the toxin's binding subunit (RTB)

The B subunit (RTB) of ricin toxin is a galactose-/N-acetyl galactosamine-specific lectin that promotes attachment and entry of ricin into host cells. RTB is also the archetype of the so-called R-type lectin family, whose members include haemagglutinins of botulinum neurotoxin (BoNT) progenitor toxins, as well as the binding subunits of cytolethal distending toxins. Although RTB is an appealing subunit vaccine candidate, as well as a potential target for immunotherapeutics, the degree to which RTB immunization elicits protective antibodies against ricin toxin remains unresolved. To address this issue, groups of mice were immunized with RTB and then challenged with 5×LD₅₀s of ricin administered intraperitoneally. Despite high RTB-specific serum antibody titers, groups of RTB immunized mice were only partially immune to ricin challenge. Analysis of a collection of RTB-specific B cell hybridomas suggested that only a small fraction of antibodies against RTB have demonstrable neutralizing activity. Two RTB-specific neutralizing monoclonal IgG₁ antibodies, 24B11 and SylH3, when passively administered to mice, were sufficient to protect the animals against a 5×LD₅₀ dose of ricin. Both 24B11 and SylH3 blocked ricin attachment to terminal galactose residues and prevented toxin binding to the surfaces of bone marrow-derived macrophages (BMM), suggesting that they function by steric hindrance and recognize epitopes located on RTB's carbohydrate recognition sub-domains (1α or 2γ). These data raise the possibility of using specific RTB sub-domains, rather than RTB itself, as antigens to more efficiently elicit neutralizing antibodies and protective immunity against ricin.     

Booster vaccination with a fractional dose of an oral cholera vaccine induces comparable vaccine-specific antibody avidity as a full dose: A randomised clinical trial

Antibody avidity is an important measure of the quality of vaccine-induced immune responses. Murine and human studies suggest that antibody avidity may be augmented by limiting access to antigen. The primary objective of this study was to evaluate in primed Swedish adults if booster vaccination with fractional doses (1/5th and 1/25th) of a model oral vaccine, the cholera vaccine Dukoral®, results in higher avidity antibody responses compared to boosting with a full vaccine dose. We also evaluated if fractional booster vaccination elicited similar magnitudes of antibody response compared to a full dose, and if the previously observed increase in antibody avidity after booster vaccination 1–2 years later occurred when boosting after a shorter interval.To this end, a randomised, open-label, exploratory Phase-II trial was performed. Swedish adults (n = 44), primed with two full doses of Dukoral®, were randomised into three groups and given a booster dose at either full (n = 14), 1/5th (n = 17) or 1/25th (n = 13) dose four months later. Antibody responses to cholera toxin B-subunit (CTB) were measured in serum and mucosal antibody in lymphocyte secretions (ALS).We found that the 1/5th and 1/25th booster doses had similar abilities as the full dose to induce significantly higher avidity anti-CTB antibody responses in both ALS and serum samples, as compared to after priming vaccination. There was a non-significant trend to lower magnitudes of ALS and serum IgA responses after the 1/5th compared to the full booster dose, and responses after the 1/25th dose were significantly lower.Our findings suggest fractional booster doses of Dukoral® four months after priming result in anti-toxoid mucosal antibody responses with increased antibody avidity compared to after priming vaccinations.ISRCTN registry identifier 11806026.     

Effectiveness of Botulinum Toxin A in Treatment of Hemiplegic Shoulder Pain: A Systematic Review and Meta-analysis

ObjectiveTo evaluate the effectiveness of botulinum toxin A (BTX-A) in the treatment of hemiplegic shoulder pain.Data SourcesPubMed, EMBASE, Elsevier, Springer, Cochrane Library, Physiotherapy Evidence Database, CNKI, and VIP were researched from the earliest records to September 1, 2020.Study SelectionRandomized controlled trials that compared shoulder BTX-A injections vs a control intervention in patients with a history of hemiplegic shoulder pain after stroke were selected. Among the 620 records screened, 9 trials with 301 eligible patients were included.Data ExtractionOutcome data were pooled according to follow-up intervals (1, 2, 4, and 12 wk). The primary evaluation indices were pain reduction (visual analog scale [VAS] score) and range of motion (ROM) improvement. The second evaluation indices were upper limb functional improvement, spasticity improvement, and incidence of adverse events. Cochrane risk-of-bias was used to assess the methodological quality of studies independently by 2 evaluators.Data SynthesisMeta-analysis revealed a statistically significant decrease in the VAS score in the BTX group vs the control group at 1, 4, and 12 weeks postinjection (wk 1: standardized mean difference [SMD], 0.91; 95% confidence interval [CI], 0.27 to 1.54; wk 4: SMD, 1.63; 95% CI, 0.76 to 2.51; wk 12: SMD, 1.96; 95% CI, 1.44 to 2.47). Furthermore, the meta-analysis demonstrated a statistically significant increase in abduction at 1, 4, and 12 weeks postinjection (wk 1: SMD, 3.71; 95% CI, 0 to 7.41; wk 4: SMD, 8.8; 95% CI, 2.22 to 15.37; wk 12: SMD, 19.59; 95% CI, 9.05 to 30.13) and external rotation at 1, 2, 4 weeks postinjection (wk 1: SMD, 5.67; 95% CI, 0.88 to 10.47; wk 2: SMD, 9.62; 95% CI, 5.57 to 13; wk 4: SMD, 6.89; 95% CI, 2.45 to 11.33) in the BTX group.ConclusionsBTX-A injection provided greater analgesic effects and increased shoulder abduction and external rotation ROM compared with steroid or placebo injection for the treatment of HSP.     

Single-dose combination nanovaccine induces both rapid and durable humoral immunity and toxin neutralizing antibody responses against Bacillus anthracis

Bacillus anthracis, the causative agent of anthrax, continues to be a prominent biological warfare and bioterrorism threat. Vaccination is likely to remain the most effective and user-friendly public health measure to counter this threat in the foreseeable future. The commercially available AVA BioThrax vaccine has a number of shortcomings where improvement would lead to a more practical and effective vaccine for use in the case of an exposure event. Identification of more effective adjuvants and novel delivery platforms is necessary to improve not only the effectiveness of the anthrax vaccine, but also enhance its shelf stability and ease-of-use. Polyanhydride particles have proven to be an effective platform at adjuvanting the vaccine-associated adaptive immune response as well as enhancing stability of encapsulated antigens. Another class of adjuvants, the STING pathway-targeting cyclic dinucleotides, have proven to be uniquely effective at inducing a beneficial inflammatory response that leads to the rapid induction of high titer antibodies post-vaccination capable of providing protection against bacterial pathogens. In this work, we evaluate the individual contributions of cyclic di-GMP (CDG), polyanhydride nanoparticles, and a combination thereof towards inducing neutralizing antibody (nAb) against the secreted protective antigen (PA) from B. anthracis. Our results show that the combination nanovaccine elicited rapid, high titer, and neutralizing IgG anti-PA antibody following single dose immunization that persisted for at least 108 DPI.     

Botulinum Toxin A and Osteosarcopenia in Experimental Animals: A Scoping Review

We conducted a scoping review to investigate the effects of intramuscular injection of Botulinum Toxin A (BoNT-A) on bone morphology. We investigated if the muscle atrophy associated with Injection of BoNT-A had effects on the neighboring bone. We used the search terms: osteopenia, bone atrophy, Botulinum Toxin A, Micro-CT, mice or rat. The following databases were searched: Medline, Embase, PubMed and the Cochrane Library, between 1990 and 2020. After removal of duplicates, 228 abstracts were identified of which 49 studies satisfied our inclusion and exclusion criteria. The majority of studies (41/49) reported a quantitative reduction in at least one measure of bone architecture based on Micro-CT. The reduction in the ratio of bone volume to tissue volume varied from 11% to 81% (mean 43%) according to the experimental set up and study time points. While longer term studies showed muscle recovery, no study showed complete recovery of all bone properties at the termination of the study. In experimental animals, intramuscular injection of BoNT-A resulted in acute muscle atrophy and acute degradation of the neighboring bone segment. These findings may have implications for clinical protocols in the use of Botulinum Toxin in children with cerebral palsy, with restraint recommended in injection protocols and consideration for monitoring bone density. Clinical studies in children with cerebral palsy receiving injections of Botulinum are indicated.