Anti‐inflammatory properties of lemon‐derived extracellular vesicles are achieved through the inhibition of ERK/NF‐κB signalling pathways

Chronic inflammation is associated with the occurrence of several diseases. However, the side effects of anti‐inflammatory drugs prompt the identification of new therapeutic strategies. Plant‐derived extracellular vesicles (PDEVs) are gaining increasing interest in the scientific community for their biological properties. We isolated PDEVs from the juice of Citrus limon L. (LEVs) and characterized their flavonoid, limonoid and lipid contents through reversed‐phase high‐performance liquid chromatography coupled to electrospray ionization quadrupole time‐of‐flight mass spectrometry (RP‐HPLC–ESI‐Q‐TOF‐MS). To investigate whether LEVs have a protective role on the inflammatory process, murine and primary human macrophages were pre‐treated with LEVs for 24 h and then were stimulated with lipopolysaccharide (LPS). We found that pre‐treatment with LEVs decreased gene and protein expression of pro‐inflammatory cytokines, such as IL‐6, IL1‐β and TNF‐α, and reduced the nuclear translocation and phosphorylation of NF‐κB in LPS‐stimulated murine macrophages. The inhibition of NF‐κB activation was associated with the reduction in ERK1‐2 phosphorylation. Furthermore, the ability of LEVs to decrease pro‐inflammatory cytokines and increase anti‐inflammatory molecules was confirmed ex vivo in human primary T lymphocytes. In conclusion, we demonstrated that LEVs exert anti‐inflammatory effects both in vitro and ex vivo by inhibiting the ERK1‐2/NF‐κB signalling pathway.     

Increased kynurenine-to-tryptophan ratio in the serum of patients infected with SARS-CoV2: An observational cohort study.

Immune dysregulation is a hallmark of patients infected by SARS-CoV2 and the balance between immune reactivity and tolerance is a key determinant of all stages of infection, including the excessive inflammatory state causing the acute respiratory distress syndrome. The kynurenine pathway (KP) of tryptophan (Trp) metabolism is activated by pro-inflammatory cytokines and drives mechanisms of immune tolerance. We examined the state of activation of the KP by measuring the Kyn:Trp ratio in the serum of healthy subjects (n = 239), and SARS-CoV2-negative (n = 305) and -positive patients (n = 89). Patients were recruited at the Emergency Room of St. Andrea Hospital (Rome, Italy). Kyn and Trp serum levels were assessed by HPLC/MS-MS. Compared to healthy controls, both SARS-CoV2-negative and -positive patients showed an increase in the Kyn:Trp ratio. The increase was larger in SARS-CoV2-positive patients, with a significant difference between SARS-CoV2-positive and -negative patients. In addition, the increase was more prominent in males, and positively correlated with age and severity of SARS-CoV2 infection, categorized as follows: 1 = no need for intensive care unit (ICU); 2 ≤ 3 weeks spent in ICU; 3 ≥ 3 weeks spent in ICU; and 4 = death. The highest Kyn:Trp values were found in SARS-CoV2-positive patients with severe lymphopenia. These findings suggest that the Kyn:Trp ratio reflects the level of inflammation associated with SARS-CoV2 infection, and, therefore, might represent a valuable biomarker for therapeutic intervention.     

The prognostic value of CD3+ tumor-infiltrating lymphocytes for stage II colon cancer according to use of adjuvant chemotherapy: A large single-institution cohort study

BackgroundHigh tumor infiltrating lymphocytes (TILs) density was previously shown to be associated with favorable prognosis for patients with colon cancer (CC). However, the impact of TILs on overall survival (OS) of stage II CC patients who received adjuvant chemotherapy (ADJ) or not (no-ADJ) is unknown. We assessed the prognostic value of CD3+ TILs in stage II CC patients according to whether they had ADJ or not.MethodsPatients treated with curative surgery for stage II CC (2002–2013) were selected from the Santa Maria alle Scotte Hospital registry. TILs at the invasive front, center of tumor, and stroma were determined by immunohistochemistry and manually quantified as the rate of TILs/total tissue areas. High TILs (H-TILs) was defined as >20%. Patients were categorized as high or low TILs (L-TILs) and ADJ or no-ADJ.ResultsOf the 678 patients included, 137 (20%) received ADJ and 541 (80%) did not. The distribution of the 4 groups were: 16% (L-TIL/ADJ), 64% (L-TIL/no-ADJ), 5% (H-TIL/ADJ), 15% (H-TIL/no-ADJ). Compared to H-TILs/no-ADJ, ADJ patients showed a significantly increased OS (P<.01) regardless of the TILs rate whereas L-TILs/no-ADJ had significantly decreased OS and higher risk of death (HR=1.41; 95% CI, 1.06–1.88; P<.0001). On multivariable analysis, the unfavorable prognostic value of L-TILs (vs. H-TILs) for no-ADJ patients was confirmed (HR=1.36; 95% CI 1.02, 1.82; P=.0373).ConclusionLow CD3+ TILs rate was associated with shorter OS in those with stage II colon cancer who did not receive adjuvant therapy. Low CD3+ TILs could be considered an additional risk factor for still ADJ-untreated stage II CC patients, which could facilitate clinical decision making.     

High serum sCD163/sTWEAK ratio is associated with lower risk of digital ulcers but more severe skin disease in patients with systemic sclerosis

Introduction

Systemic sclerosis (SSc) is an autoimmune disease characterized by chronic inflammation, vascular injury and excessive fibrosis. CD163 is a scavenger receptor which affects inflammatory response and may contribute to connective tissue remodelling. It has recently been demonstrated that CD163 can bind and neutralize the TNF-like weak inducer of apoptosis (TWEAK), a multifunctional cytokine which regulates inflammation, angiogenesis and tissue remodelling. We aimed to investigate the relationships between serum levels of soluble CD163 (sCD163) and soluble TWEAK (sTWEAK) in relation to disease manifestations in SSc patients.

Methods

This study included 89 patients with SSc who had not received immunosuppressive drugs or steroids for at least 6 months and 48 age- and sex-matched healthy controls (HC) from four European centres. Serum concentrations of sTWEAK and sCD163 were measured using commercially available ELISA kits.

Results

The mean serum concentrations of sTWEAK were comparable between SSc patients (mean +/- SD: 270 +/- 171 pg/mL) and HC (294 +/- 147pg/mL, P >0.05). Concentration of sCD163 and sCD163/sTWEAK ratio were significantly greater in SSc patients (984 +/- 420 ng/mL and 4837 +/- 3103, respectively) as compared to HC (823 +/- 331 ng/mL and 3115 +/- 1346 respectively, P <0.05 for both). High sCD163 levels and a high sCD163/sTWEAK ratio (defined as > mean +2SD of HC) were both associated with a lower risk of digital ulcers in SSc patients (OR, 95%CI: 0.09; 0.01, 0.71, and 0.17; 0.06, 0.51, respectively). Accordingly, patients without digital ulcers had a significantly higher sCD163 concentration and sCD163/sTWEAK ratio as compared to SSc patients with digital ulcers (P <0.01 for both) and HC (P <0.05 for both). A high sCD163/sTWEAK ratio, but not high sCD163 levels, was associated with greater skin involvement.

Conclusions

The results of our study indicate that CD163-TWEAK interactions might play a role in the pathogenesis of SSc and that CD163 may protect against the development of digital ulcers in SSc. Further studies are required to reveal whether targeting of the CD163-TWEAK pathway might be a potential strategy for treating vascular disease and/or skin fibrosis in SSc.     

Systemic sclerosis sera affect fibrillin-1 deposition by dermal blood microvascular endothelial cells: therapeutic implications of cyclophosphamide

Introduction

Systemic sclerosis (SSc) is a connective tissue disorder characterized by endothelial cell injury, autoimmunity and fibrosis. The following three fibrillin-1 alterations have been reported in SSc. (1) Fibrillin-1 microfibrils are disorganized in SSc dermis. (2) Fibrillin-1 microfibrils produced by SSc fibroblasts are unstable. (3) Mutations in the FBN1 gene and anti-fibrillin-1 autoantibodies have been reported in SSc. Fibrillin-1 microfibrils, which are abundantly produced by blood and lymphatic microvascular endothelial cells (B-MVECs and Ly-MVECs, respectively), sequester in the extracellular matrix the latent form of the potent profibrotic cytokine transforming growth factor β (TGF-β). In the present study, we evaluated the effects of SSc sera on the deposition of fibrillin-1 and microfibril-associated glycoprotein 1 (MAGP-1) and the expression of focal adhesion molecules by dermal B-MVECs and Ly-MVECs.

Methods

Dermal B-MVECs and Ly-MVECs were challenged with sera from SSc patients who were treatment-naïve or under cyclophosphamide (CYC) treatment and with sera from healthy controls. Fibrillin-1/MAGP-1 synthesis and deposition and the expression of α_vβ₃ integrin/phosphorylated focal adhesion kinase and vinculin/actin were evaluated by immunofluorescence and quantified by morphometric analysis.

Results

Fibrillin-1 and MAGP-1 colocalized in all experimental conditions, forming a honeycomb pattern in B-MVECs and a dense mesh of short segments in Ly-MVECs. In B-MVECs, fibrillin-1/MAGP-1 production and α_vβ₃ integrin expression significantly decreased upon challenge with sera from naïve SSc patients compared with healthy controls. Upon challenge of B-MVECs with sera from CYC-treated SSc patients, fibrillin-1/MAGP-1 and α_vβ₃ integrin levels were comparable to those of cells treated with healthy sera. Ly-MVECs challenged with SSc sera did not differ from those treated with healthy control sera in the expression of any of the molecules assayed.

Conclusions

Because of the critical role of fibrillin-1 in sequestering the latent form of TGF-β in the extracellular matrix, its decreased deposition by B-MVECs challenged with SSc sera might contribute to dermal fibrosis. In SSc, CYC treatment might limit fibrosis through the maintenance of physiologic fibrillin-1 synthesis and deposition by B-MVECs.     

Interkingdom Complementation Reveals Structural Conservation and Functional Divergence of 14-3-3 Proteins

The 14-3-3s are small acidic cytosolic proteins that interact with multiple clients and participate in essential cellular functions in all eukaryotes. Available structural and functional information about 14-3-3s is largely derived from higher eukaryotes, which contain multiple members of this protein family suggesting functional specialization. The exceptional sequence conservation among 14-3-3 family members from diverse species suggests a common ancestor for 14-3-3s, proposed to have been similar to modern 14-3-3ε isoforms. Structural features of the sole family member from the protozoan Giardia duodenalis (g14-3-3), are consistent with this hypothesis, but whether g14-3-3 is functionally homologous to the epsilon isoforms is unknown. We use inter-kingdom reciprocal functional complementation and biochemical methods to determine whether g14-3-3 is structurally and functionally homologous with members of the two 14-3-3 conservation groups of the metazoan Drosophila melanogaster. Our results indicate that although g14-3-3 is structurally homologous to D14-3-3ε, functionally it diverges presenting characteristics of other 14-3-3s. Given the basal position of Giardia in eukaryotic evolution, this finding is consistent with the hypothesis that 14-3-3ε isoforms are ancestral to other family members.     

Syndromic Algorithms for Detection of Gambiense Human African Trypanosomiasis in South Sudan

Background

Active screening by mobile teams is considered the best method for detecting human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense but the current funding context in many post-conflict countries limits this approach. As an alternative, non-specialist health care workers (HCWs) in peripheral health facilities could be trained to identify potential cases who need testing based on their symptoms. We explored the predictive value of syndromic referral algorithms to identify symptomatic cases of HAT among a treatment-seeking population in Nimule, South Sudan.

Methodology/Principal Findings

Symptom data from 462 patients (27 cases) presenting for a HAT test via passive screening over a 7 month period were collected to construct and evaluate over 14,000 four item syndromic algorithms considered simple enough to be used by peripheral HCWs. For comparison, algorithms developed in other settings were also tested on our data, and a panel of expert HAT clinicians were asked to make referral decisions based on the symptom dataset. The best performing algorithms consisted of three core symptoms (sleep problems, neurological problems and weight loss), with or without a history of oedema, cervical adenopathy or proximity to livestock. They had a sensitivity of 88.9–92.6%, a negative predictive value of up to 98.8% and a positive predictive value in this context of 8.4–8.7%. In terms of sensitivity, these out-performed more complex algorithms identified in other studies, as well as the expert panel. The best-performing algorithm is predicted to identify about 9/10 treatment-seeking HAT cases, though only 1/10 patients referred would test positive.

Conclusions/Significance

In the absence of regular active screening, improving referrals of HAT patients through other means is essential. Systematic use of syndromic algorithms by peripheral HCWs has the potential to increase case detection and would increase their participation in HAT programmes. The algorithms proposed here, though promising, should be validated elsewhere.     

Bisphenol-A Impairs Insulin Action and Up-Regulates Inflammatory Pathways in Human Subcutaneous Adipocytes and 3T3-L1 Cells

Current evidence indicates that chemical pollutants may interfere with the homeostatic control of nutrient metabolism, thereby contributing to the increased prevalence of metabolic disorders. Bisphenol-A (BPA) is a lipophilic compound contained in plastic which is considered a candidate for impairing energy and glucose metabolism. We have investigated the impact of low doses of BPA on adipocyte metabolic functions. Human adipocytes derived from subcutaneous adipose tissue and differentiated 3T3-L1 cells were incubated with BPA, in order to evaluate the effect on glucose utilization, insulin sensitivity and cytokine secretion. Treatment with 1nM BPA significantly inhibited insulin-stimulated glucose utilization, without grossly interfering with adipocyte differentiation. Accordingly, mRNA levels of the adipogenic markers PPARγ and GLUT4 were unchanged upon BPA exposure. BPA treatment also impaired insulin-activated receptor phosphorylation and signaling. Moreover, adipocyte incubation with BPA was accompanied by increased release of IL-6 and IFN-γ, as assessed by multiplex ELISA assays, and by activation of JNK, STAT3 and NFkB pathways. Treatment of the cells with the JNK inhibitor SP600125 almost fully reverted BPA effect on insulin signaling and glucose utilization. In conclusion, low doses of BPA interfere with inflammatory/insulin signaling pathways, leading to impairment of adipose cell function.     

From the cradle of grapevine domestication: molecular overview and description of Georgian grapevine (Vitis vinifera L.) germplasm

Historical information and archaeological and palaeobotanical findings point Georgia, in the South Caucasus, as a cradle for grapevine (Vitis vinifera L.) domestication from its wild form (V. vinifera silvestris Beck.) and subsequent selection and development of varieties with characters suitable for human consumption. The hypothesis of Georgia being a center of domestication, combined with its distance from western countries and the importance of its viticulture and wine production, make Georgian grape germplasm particularly interesting to be investigated under the genetic point of view. Twenty nuclear microsatellite loci were used to genotype 112 Georgian grapevine accessions (V. vinifera sativa Beck.) from germplasm collections and 18 from spontaneous growing plants (V. vinifera silvestris Beck.) found in wild conditions and to compare them to a large international cultivar collection in France. Data analysis shows that Georgian grapevine germplasm has maintained distinctive traits despite arrival of international, foreign varieties and still conserve characteristics of local breeding linked to traditional wine production regions of the country. Results have identified alleles, overall loci, well represented in the Georgian germplasm (cultivated and wild) and absent or poorly represented in other countries, highlighting uniqueness and originality of traits of this viticulture. Moreover, the search for relationships between Georgian and foreign viticulture has evidenced few interesting cases linking the Georgian varieties with Western European ones and with neighboring Caucasian countries, helping to identify the real place of origin in some doubtful cases. In addition, populations or sparse individuals of wild grapevine still preserved in the Georgian natural environments present smaller genetic distances with local cultivars than in other European regions. Principal component analysis (PCA) has also identified special overlapping of the wild compartment with some cultivated varieties. This work provides a highly significant new contribution to applied aspects of Georgian grapevine genetic resources management and use. Uniqueness of the Georgian cultivated grapevine gene pool together with its close relatedness with the wild compartment makes this country a good candidate to address questions regarding domestication and grapevine genetic resource conservation.     

A Re-Emerging Marker for Prognosis in Hepatocellular Carcinoma: The Add-Value of FISHing c-myc Gene for Early Relapse

Hepatocellular carcinoma is one leading cause of cancer-related death and surgical resection is still one of the major curative therapies. Recently, there has been a major effort to find mechanisms involved in carcinogenesis and early relapse. c-myc gene abnormality is found in hepatocarcinogenesis. Our aim was to analyze the role of c-myc as prognostic factor in terms of overall survival and disease-free survival and to investigate if c-myc may be an important target for therapy. We studied sixty-five hepatocellular carcinomas submitted to surgical resection with curative intent. Size, macro-microvascular invasion, necrosis, number of nodules, grading and serum alfa-fetoprotein level were registered for all cases. We evaluated the c-myc aberrations by using break-apart FISH probes. Probes specific for the centromeric part of chromosome 8 and for the locus specific c-myc gene (8q24) were used to assess disomy, gains of chromosomes (polysomy due to polyploidy) and amplification. c-myc gene amplification was scored as 8q24/CEP8 > 2. Statistical analysis for disease-free survival and overall survival were performed. At molecular level, c-myc was amplified in 19% of hepatocellular carcinoma, whereas showed gains in 55% and set wild in 26% of cases. The 1- and 3-year disease-free survival and overall survival for disomic, polysomic and amplified groups were significantly different (p=0.020 and p=.018 respectively). Multivariate analysis verified that the AFP and c-myc status (amplified vs. not amplified) were significant prognostic factors for overall patients survival. c-myc gene amplification is significantly correlated with disease-free survival and overall survival in patients with hepatocellular carcinoma after surgical resection and this model identifies patients with risk of early relapse (≤12 months). We suggest that c-myc assessment may be introduced in the clinical practice for improving prognostication (high and low risk of relapse) routinely and may have be proposed as biomarker of efficacy to anti-c-myc targeted drugs in clinical trials.