Effect of low-level laser therapy on the expression of inflammatory mediators and on neutrophils and macrophages in acute joint inflammation

Introduction

Inflammation of the synovial membrane plays an important role in the pathophysiology of osteoarthritis (OA). The synovial tissue of patients with initial OA is characterized by infiltration of mononuclear cells and production of proinflammatory cytokines and other mediators of joint injury. The objective was to evaluate the effect of low-level laser therapy (LLLT) operating at 50 mW and 100 mW on joint inflammation in rats induced by papain, through histopathological analysis, differential counts of inflammatory cells (macrophages and neutrophils), as well as gene expression of interleukin 1-beta and 6 (IL-1β and IL-6), and protein expression of tumor necrosis factor alpha (TNFα).

Methods

Male Wistar rats (n = 60) were randomly divided into four groups of 15 animals, namely: a negative control group; an inflammation injury positive control group; a 50 mW LLLT group, subjected to injury and treated with 50 mW LLLT; and a 100 mW LLLT group, subjected to injury and treated with 100 mW LLLT. The animals were subject to joint inflammation (papain solution, 4%) and then treated with LLLT (808 nm, 4 J, 142.4 J/cm², spot size 0.028 for both groups). On the day of euthanasia, articular lavage was collected and immediately centrifuged; the supernatant was saved for analysis of expression of TNFα protein by enzyme-linked immunosorbent assay and expression of IL-1β and IL-6 mRNA by real-time polymerase chain reaction. A histologic examination of joint tissue was also performed. For the statistical analysis, analysis of variance with Tukey''s post-hoc test was used for comparisons between each group. All data are expressed as mean values and standard deviation, with P < 0.05.

Results

Laser treatment with 50 mW was more efficient than 100 mW in reducing cellular inflammation, and decreased the expression of IL-1β and IL-6. However, the 100 mW treatment led to a higher reduction of TNFα compared with the 50 mW treatment.

Conclusions

LLLT with 50 mW was more efficient in modulating inflammatory mediators (IL-1β, IL-6) and inflammatory cells (macrophages and neutrophils), which correlated with the histology that showed a reduction in the inflammatory process.     

Molecular model of hexokinase binding to the outer mitochondrial membrane porin (VDAC1): Implication for the design of new cancer therapies

A key feature of many cancers is the capacity and the propensity to metabolize glucose to lactic acid at a very high rate even in the presence of oxygen. This characteristic was first discovered in 1924 by Otto Heinrich Warburg. Hexokinase, the first enzyme in the glycolytic pathway, not only improves the cell's energy supply in malignant cells, but also protects cancer cells against apoptosis through direct interaction with mitochondria and with the Voltage Dependent Anion Channel 1 (VDAC1). The rupture of HK:VDAC1 protein complex provides a therapeutic opportunity, as this association appears to protect tumor cells from mitochondrial outer membrane permeabilization, an event that marks the point of no return in multiple pathways leading to cell death. In the absence of a crystallographic structure and in order to perform an in silico screening of possible small molecules able to inhibit the protein association, we are presenting a computational model of HK-I:VDAC1 complex. It appears as evident how the first 15 N-terminal residues of HK-I interact with the inner part of the barrel of VDAC1 and not with the outside walls, within the mitochondrial membrane as previously believed. This finding is in agreement with the existence of a secondary ATP binding site in the same N-terminal region of HK-I which seems to have a crucial role in HK-I interaction with VDAC1. This evidence appears to be in accord also with the high levels of ATP that are found in cancer cells. Eventually such arrangements may contribute to stabilize the tertiary structure of VDAC1 while shielding from pro-apoptotic factor binding, protecting in a synergic way the tumoral cell from programmed death.     

E-NTPDase and E-ADA activities in lymphocytes associated with the immune response of rats experimentally infected with Toxoplasma gondii

An investigation of E-NTPDase and E-ADA activities in lymphocytes from rats experimentally infected with Toxoplasma gondii was carried out in this study. For this purpose, twenty four adult male Wistar rats were divided in two groups/four subgroups (A1 and A2; B1 and B2–6 animal/each group), with “A” as uninfected and “B” inoculated with T. gondii (RH strain). Sampling was performed on days 5 and 10 post-infection (p.i.), with evaluation of hemogram, immunoglobulins (IgM and IgG) and activity of E-NTPDase and E-ADA in lymphocytes. Enzymes essays showed ATP hydrolysis increased on days 5 (P < 0.05) and 10 (P < 0.01) p.i., as well as an increase of ADP hydrolysis on day 10 (P < 0.01) p.i. E-ADA activity on lymphocytes was also increased in both evaluated periods (P < 0.01). Based on E-NTPDase and E-ADA increased activities observed on lymphocytes, it is possible to suggest their involvement in an anti-inflammatory response, consisting of a modulatory response, preventing excessive tissue damage caused by the infection with Toxoplasma gondii.     

Reproduction and monogamy in captive flock of greater flamingos (Phoenicopterus Roseus)

Due to the great number of greater flamingos in captivity and their long life span, studying their behavior and welfare might be useful to improve the husbandry and breeding of this species in zoos. This study aimed to investigate factors affecting the breeding activity of captive greater flamingos (Phoenicopterus roseus) from 2012 to 2016. To estimate reproductive success, the number of pairs, eggs laid, and hatchlings were recorded. In addition, information on age, egg-laying history of the partners, and pair composition was collected. An increase in the number of pairs, eggs, and hatchlings was reported over the years. For each breeding season, there were pairs who laid more than once, especially in 2014 and 2015. Approximately 50% of pairs were monogamous between consecutive years; however, the percentage dropped gradually when comparing nonconsecutive years. Senescence and previous experience seemed to affect the reproductive success of the study flamingos. In conclusion, different factors can influence the reproduction of greater flamingos in zoos. All these factors are related to nonhuman animal welfare and need to be considered in developing and improving management practices.     

Building a baseline for habitat-forming corals by a multi-source approach,including Web Ecological Knowledge

In the Mediterranean, habitat-forming corals often characterize essential fish habitats. While their distribution is sufficiently known for the western basin, few data are available from the Central-Eastern Mediterranean Sea (CEM). This study fills this gap supplying the largest dataset ever built on the geographical and bathymetric distribution of the most relevant habitat-forming corals (Eunicella cavolini, Eunicella verrucosa, Eunicella singularis, Leptogorgia sarmentosa, Paramuricea clavata, Corallium rubrum and Savalia savaglia) of the CEM. Information collected from different sources such as literature, citizen science, and from the World Wide Web (WWW) was combined. Videos published on the WWW provided additional information on the presence of fishing lines and signs of damage, as well as on the distribution of purple and yellow-purple colonies of Paramuricea clavata. The study highlighted the impressive amount of information that the WWW can offer to scientists, termed here as Web Ecological Knowledge (WEK). The WEK is constantly fuelled by internauts, representing a free, refreshable, long-term exploitable reservoir of information. A quick and easy method to retrieve data from the WWW was illustrated. In addition, the distribution of corals was overlapped to marine protected areas and to the distribution of environmental conditions suitable for coralligenous habitats, fragile biogenic Mediterranean structures hosting complex assemblages in need of strict protection. The collected data allowed identifying priority areas with high species diversity and sites that are impacted by fishing activities. Supplied data can correctly address conservation and restoration policies in the CEM, adding an important contribution to ecosystem-based marine spatial planning.     

Genomic analysis reveals depression due to both individual and maternal inbreeding in a free‐living mammal population

There is ample evidence for inbreeding depression manifested as a reduction in fitness or fitness‐related traits in the focal individual. In many organisms, fitness is not only affected by genes carried by the individual, but also by genes carried by their parents, for example if receiving parental care. While maternal effects have been described in many systems, the extent to which inbreeding affects fitness directly through the focal individual, or indirectly through the inbreeding coefficients of its parents, has rarely been examined jointly. The Soay sheep study population is an excellent system in which to test for both effects, as lambs receive extended maternal care. Here, we tested for both maternal and individual inbreeding depression in three fitness‐related traits (birthweight and weight and hindleg length at 4 months of age) and three fitness components (first‐year survival, adult annual survival and annual breeding success), using either pedigree‐derived inbreeding or genomic estimators calculated using ~37 000 SNP markers. We found evidence for inbreeding depression in 4‐month hindleg and weight, first‐year survival in males, and annual survival and breeding success in adults. Maternal inbreeding was found to depress both birthweight and 4‐month weight. We detected more instances of significant inbreeding depression using genomic estimators than the pedigree, which is partly explained through the increased sample sizes available. In conclusion, our results highlight that cross‐generational inbreeding effects warrant further exploration in species with parental care and that modern genomic tools can be used successfully instead of, or alongside, pedigrees in natural populations.     

CRL3IBTK Regulates the Tumor Suppressor Pdcd4 through Ubiquitylation Coupled to Proteasomal Degradation

The human inhibitor of Bruton''s tyrosine kinase isoform α (IBtkα) is a BTB protein encoded by the IBTK gene, which maps to chromosomal locus 6q14.1, a mutational hot spot in lymphoproliferative disorders. Here, we demonstrate that IBtkα forms a CRL3^IBTK complex promoting its self-ubiquitylation. We identified the tumor suppressor Pdcd4 as IBtkα interactor and ubiquitylation substrate of CRL3^IBTK for proteasomal degradation. Serum-induced degradation of Pdcd4 required both IBtkα and Cul3, indicating that CRL3^IBTK regulated the Pdcd4 stability in serum signaling. By promoting Pdcd4 degradation, IBtkα counteracted the suppressive effect of Pdcd4 on translation of reporter luciferase mRNAs with stem-loop structured or unstructured 5′-UTR. IBtkα depletion by RNAi caused Pdcd4 accumulation and decreased the translation of Bcl-xL mRNA, a well known target of Pdcd4 repression. By characterizing CRL3^IBTK as a novel ubiquitin ligase, this study provides new insights into regulatory mechanisms of cellular pathways, such as the Pdcd4-dependent translation of mRNAs.     

Pneumococcal polysaccharide vaccination in adults undergoing immunosuppressive treatment for inflammatory diseases – a longitudinal study

IntroductionPatients undergoing immunosuppressive therapy are at increased risk of infection. Community-acquired pneumonia and invasive pneumococcal disease account for substantial morbidity and mortality in this population and may be prevented by vaccination. Ideally, immunization to pneumococcal antigens should take place before the start of immunosuppressive treatment. Often, however, the treatment cannot be delayed. Little is known about the efficacy of pneumococcal vaccines during immunosuppressive treatment. The objectives of this study were to determine the percentage of vaccine-naïve, immunosuppressed adults with inflammatory diseases seroprotected against Streptococcus pneumoniae and to assess factors associated with the immunogenicity, clinical impact and safety of 23-valent pneumococcal polysaccharide vaccine (PPV) in seronegative subjects.MethodsThis observational study included patients 18 years of age and older who were receiving prednisone ≥20 mg/day or other immunosuppressive drugs. Exclusion criteria were PPV administration in the previous 5 years, intravenous immunoglobulins and pregnancy. Serum immunoglobulin G (IgG) antibody levels against six pneumococcal serotypes were measured. Seropositivity was defined as IgG of 0.5 μg/ml or greater for at least four of six serotypes. Seronegative patients received PPV, and seropositive patients were included as a comparison group. Vaccine response and tolerance were assessed after 4–8 weeks. Disease activity was evaluated on the basis of the Physician Global Assessment scores. Serology was repeated after 1 year, and information on any kind of infection needing medical attention was collected. Outcomes were the proportion of seropositivity and infections between vaccinated and unvaccinated patients.ResultsOf 201 included patients, 35 received high-dose corticosteroids and 181 were given immunosuppressive drugs. Baseline seronegativity in 60 (30 %) patients was associated with corticotherapy and lower total IgG. After PPV, disease activity remained unchanged or decreased in 81 % of patients, and 87 % became seropositive. After 1 year, 67 % of vaccinated compared with 90 % of observed patients were seropositive (p < 0.001), whereas the rate of infections did not differ between groups. Those still taking prednisone ≥10 mg/day tended to have poorer serological responses and had significantly more infections.ConclusionsPPV was safe and moderately effective based on serological response. Seropositivity to pneumococcal antigens significantly reduced the risk of infections. Sustained high-dose corticosteroids were associated with poor vaccine response and more infections.     

Biological and Structural Characterization of Crotoxin and New Isoform of Crotoxin B PLA<Subscript>2</Subscript> (F6a) from <Emphasis Type="Italic">Crotalus durissus collilineatus</Emphasis> Snake Venom

A new crotoxin B isoform PLA₂ (F6a), from Crotalus durissus collilineatus was purified from by one step reverse phase HPLC chromatography using μ-Bondapack C-18 column analytic. The new crotoxin B isoform PLA₂ (F6a), complex crotoxin, the catalytic subunit crotoxin B isoform PLA₂ (F6a) and two crotapotin isoforms (F3 and F4), were isolated from the venom of Crotalus durissus collilineatus. The crotapotins isoforms F3 and F4 had similar chemical properties, the two proteins different in their ability to inhibit of isoforms of PLA₂ (F6 and F6a). The molecular masses estimated by MALDI-TOF mass spectrometry were: crotoxin B: 14,943.14 Da, crotapotin F3: 8,693.24 Da, and crotapotin F4: 9 314.56 Da. The new crotoxin B isoform PLA₂ (F6a) contained 122 amino acid residues and a pI of 8.58. Its amino acid sequence presents high identity with those of other PLA₂s, particularly in the calcium binding loop and active site helix 3. It also presents similarities in the C-terminal region with other myotoxic PLA₂s. The new crotoxin B isoform PLA₂ (F6a) contained 122 amino acid residues, with a primary structure of HLLQFNKMIK FETRRNAIPP YAFYGCYCGW GGRGRPKDAT DRCCFVHDCC YGKLAKCNTK WDFYRYSLKS GYITCGKGTW CEEQICECDR VAAECLRRSL STYRYGYMIY PDSRCRGPSE TC. A neuromuscular blocking activity was induced by crotoxin and new crotoxin B isoform PLA₂ (F6a) in the isolated mouse phrenic nerve diaphragm and the biventer cervicis chick nerve-muscle preparation. Whole crotoxin was devoid of cytolytic activity upon myoblasts and myotubes in vitro, whereas new crotoxin B isoform PLA₂ (F6a) was clearly cytotoxic to these cells.