Feasibility and efficacy of combined pancreatic islet-lung transplantation in cystic fibrosis-related diabetes-PIM study: A multicenter phase 1-2 trial.

Cystic fibrosis-related diabetes (CFRD) is a common complication of cystic fibrosis (CF), and restoring metabolic control in these patients may improve their management after lung transplantation. In this multicenter, prospective, phase 1-2 trial, we evaluate the feasibility and metabolic efficacy of combined pancreatic islet-lung transplantation from a single donor in patients with CFRD, terminal respiratory failure, and poorly controlled diabetes. Islets were infused via the portal vein under local anesthesia, 1 week after lung transplantation. At 1 year, the primary outcome was transplant success as evaluated by a composite score including four parameters (weight, fasting glycemia, HbA1c, and insulin requirements). Ten participants (age: 24 years [17-31], diabetes duration: 8 years [4-12]) received a combined islet-lung transplant with 2892 IEQ/kg [2293-6185]. Transplant success was achieved in 7 out of 10 participants at 1-year post transplant. Fasting plasma C-peptide increased from 0.91 µg/L [0.56-1.29] to 1.15 µg/L [0.77-2.2], HbA1c decreased from 7.8% [6.5-8.3] (62 mmol/mol [48-67]) to 6.7% [5.5-8.0] (50 mmol/mol [37-64]), with 38% decrease in daily insulin doses. No complications related to the islet injection procedure were reported. In this pilot study, combined pancreatic islet-lung transplantation restored satisfactory metabolic control and pulmonary function in patients with CF, without increasing the morbidity of lung transplantation.

[Total pancreatectomy and islet auto-transplantation: current state and new perspectives]. / Pancréatectomie totale et autotransplantation d'îlots†: état actuel et nouvelles perspectives.

Total pancreatectomy is a procedure primarily performed for chronic pancreatitis refractory to conservative therapy. It may nevertheless be indicated in the event of a malignant tumor, either as a treatment for a surgical complication or as a prevention of anastomotic leakage. If possible, islet auto-transplantation should be combined with total pancreatectomy for benign disease, in order to prevent a severe diabetes. Until recently, malignant disease was considered an absolute contraindication to islet auto-transplantation. A recent series from Milan showed promising oncological results in auto-transplantation for malignant disease, opening up new perspectives for total pancreatectomy for cancer.

La pancréatectomie totale est une procédure principalement effectuée pour une pancréatite chronique réfractaire au traitement conservateur. Elle peut néanmoins être indiquée en cas de tumeur maligne, soit comme traitement d'une complication chirurgicale, soit en prévention de fuite anastomotique. Dans la mesure du possible, une autogreffe d'îlots de Langerhans devrait être associée à une pancréatectomie totale pour maladie bénigne, dans le but de prévenir un diabète pancréatoprive. Jusqu'à récemment, une pathologie maligne était considérée comme une contre-indication absolue à une autogreffe d'îlots. Une série récente de Milan a montré des résultats oncologiques prometteurs en cas d'autogreffe pour pathologies malignes, ouvrant de nouvelles perspectives à la pancréatectomie totale pour cancer.

Bio-Engineering of Pre-Vascularized Islet Organoids for the Treatment of Type 1 Diabetes.

Lack of rapid revascularization and inflammatory attacks at the site of transplantation contribute to impaired islet engraftment and suboptimal metabolic control after clinical islet transplantation. In order to overcome these limitations and enhance engraftment and revascularization, we have generated and transplanted pre-vascularized insulin-secreting organoids composed of rat islet cells, human amniotic epithelial cells (hAECs), and human umbilical vein endothelial cells (HUVECs). Our study demonstrates that pre-vascularized islet organoids exhibit enhanced in vitro function compared to native islets, and, most importantly, better engraftment and improved vascularization in vivo in a murine model. This is mainly due to cross-talk between hAECs, HUVECs and islet cells, mediated by the upregulation of genes promoting angiogenesis (vegf-a) and ß cell function (glp-1r, pdx1). The possibility of adding a selected source of endothelial cells for the neo-vascularization of insulin-scereting grafts may also allow implementation of ß cell replacement therapies in more favourable transplantation sites than the liver.

Flow Cytometry Analysis of Circulating Extracellular Vesicle Subtypes from Fresh Peripheral Blood Samples.

Extracellular vesicles (EVs) are released by shedding during different physiological processes and are increasingly thought to be new potential biomarkers. However, the impact of pre-analytical processing phases on the final measurement is not predictable and for this reason, the translation of basic research into clinical practice has been precluded. Here we have optimized a simple procedure in combination with polychromatic flow cytometry (PFC), to identify, classify, enumerate, and separate circulating EVs from different cell origins. This protocol takes advantage of a lipophilic cationic dye (LCD) able to probe EVs. Moreover, the application of the newly optimized PFC protocol here described allowed the obtainment of repeatable EVs counts. The translation of this PFC protocol to fluorescence-activated cell sorting allowed us to separate EVs from fresh peripheral blood samples. Sorted EVs preparations resulted particularly suitable for proteomic analyses, which we applied to study their protein cargo. Here we show that LCD staining allowed PFC detection and sorting of EVs from fresh body fluids, avoiding pre-analytical steps of enrichment that could impact final results. Therefore, LCD staining is an essential step towards the assessment of EVs clinical significance.

Generation of insulin-secreting organoids: a step toward engineering and transplanting the bioartificial pancreas.

Diabetes is a major health issue of increasing prevalence. ß-cell replacement, by pancreas or islet transplantation, is the only long-term curative option for patients with insulin-dependent diabetes. Despite good functional results, pancreas transplantation remains a major surgery with potentially severe complications. Islet transplantation is a minimally invasive alternative that can widen the indications in view of its lower morbidity. However, the islet isolation procedure disrupts their vasculature and connection to the surrounding extracellular matrix, exposing them to ischemia and anoikis. Implanted islets are also the target of innate and adaptive immune attacks, thus preventing robust engraftment and prolonged full function. Generation of organoids, defined as functional 3D structures assembled with cell types from different sources, is a strategy increasingly used in regenerative medicine for tissue replacement or repair, in a variety of inflammatory or degenerative disorders. Applied to ß-cell replacement, it offers the possibility to control the size and composition of islet-like structures (pseudo-islets), and to include cells with anti-inflammatory or immunomodulatory properties. In this review, we will present approaches to generate islet cell organoids and discuss how these strategies can be applied to the generation of a bioartificial pancreas for the treatment of type 1 diabetes.

Pest categorisation of non-EU viruses of Rubus L.

The Panel on Plant Health of EFSA conducted a pest categorisation of 17 viruses of Rubus L. that were previously classified as either non-EU or of undetermined standing in a previous opinion. These infectious agents belong to different genera and are heterogeneous in their biology. Blackberry virus X, blackberry virus Z and wineberry latent virus were not categorised because of lack of information while grapevine red blotch virus was excluded because it does not infect Rubus. All 17 viruses are efficiently transmitted by vegetative propagation, with plants for planting representing the major pathway for entry and spread. For some viruses, additional pathway(s) are Rubus seeds, pollen and/or vector(s). Most of the viruses categorised here infect only one or few plant genera, but some of them have a wide host range, thus extending the possible entry pathways. Cherry rasp leaf virus, raspberry latent virus, raspberry leaf curl virus, strawberry necrotic shock virus, tobacco ringspot virus and tomato ringspot virus meet all the criteria to qualify as potential Union quarantine pests (QPs). With the exception of impact in the EU territory, on which the Panel was unable to conclude, blackberry chlorotic ringspot virus, blackberry leaf mottle-associated virus, blackberry vein banding-associated virus, blackberry virus E, blackberry virus F, blackberry virus S, blackberry virus Y and blackberry yellow vein-associated virus satisfy all the other criteria to be considered as potential QPs. Black raspberry cryptic virus, blackberry calico virus and Rubus canadensis virus 1 do not meet the criterion of having a potential negative impact in the EU. For several viruses, the categorisation is associated with high uncertainties, mainly because of the absence of data on biology, distribution and impact. Since the opinion addresses non-EU viruses, they do not meet the criteria to qualify as potential Union regulated non-quarantine pests.

Differential response to hepatic differentiation stimuli of amniotic epithelial cells isolated from four regions of the amniotic membrane.

Human Amniotic Epithelial Cells (hAEC) isolated from term placenta are a promising source for regenerative medicine. However, it has long been debated whether the hAEC population consists of heterogeneous or homogeneous cells. In a previous study, we investigated the characteristics of hAEC isolated from four different regions of the amniotic membrane finding significant heterogeneity. The aim of this study was to evaluate the hepatic differentiation capability of hAEC isolated from these four regions. Human term placentae were collected after caesarean section and hAEC were isolated from four regions of the amniotic membrane (R1-R4, according to their relative distance from the umbilical cord) and treated in hepatic differentiation conditions for 14 days. hAEC-derived hepatocyte-like cells showed marked differences in the expression of hepatic markers: R4 showed higher levels of Albumin and Hepatocyte Nuclear Factor (HNF) 4α whereas R1 expressed higher Cytochrome P450 enzymes, both at the gene and protein level. These preliminary results suggest that hAEC isolated from R1 and R4 of the amniotic membrane are more prone to hepatic differentiation. Therefore, the use of hAEC from a specific region of the amniotic membrane should be taken into consideration as it could have an impact on the outcome of therapeutic applications.

Shielding islets with human amniotic epithelial cells enhances islet engraftment and revascularization in a murine diabetes model.

Hypoxia is a major cause of considerable islet loss during the early posttransplant period. Here, we investigate whether shielding islets with human amniotic epithelial cells (hAECs), which possess anti-inflammatory and regenerative properties, improves islet engraftment and survival. Shielded islets were generated on agarose microwells by mixing rat islets (RIs) or human islets (HI) and hAECs (100 hAECs/IEQ). Islet secretory function and viability were assessed after culture in hypoxia (1% O2 ) or normoxia (21% O2 ) in vitro. In vivo function was evaluated after transplant under the kidney capsule of diabetic immunodeficient mice. Graft morphology and vascularization were evaluated by immunohistochemistry. Both shielded RIs and HIs show higher viability and increased glucose-stimulated insulin secretion after exposure to hypoxia in vitro compared with control islets. Transplant of shielded islets results in considerably earlier normoglycemia and vascularization, an enhanced glucose tolerance, and a higher ß cell mass. Our results show that hAECs have a clear cytoprotective effect against hypoxic damages in vitro. This strategy improves ß cell mass engraftment and islet revascularization, leading to an improved capacity of islets to reverse hyperglycemia, and could be rapidly applicable in the clinical situation seeing that the modification to HIs are minor.

Spittlebugs of Mediterranean Olive Groves: Host-Plant Exploitation throughout the Year.

Spittlebugs are the vectors of the bacterium Xylella fastidiosa Wells in Europe, the causal agent of olive dieback epidemic in Apulia, Italy. Selection and distribution of different spittlebug species on host-plants were investigated during field surveys in 2016-2018 in four olive orchards of Apulia and Liguria Regions of Italy. The nymphal population in the herbaceous cover was estimated using quadrat samplings. Adults were collected by sweeping net on three different vegetational components: herbaceous cover, olive canopy, and wild woody plants. Three species of spittlebugs were collected: Philaenus spumarius L., Neophilaenus campestris (Fallén), and Aphrophora alni (L.) (Hemiptera: Aphrophoridae). Philaenus spumarius was the predominant species both in Apulia and Liguria olive groves. Nymphal stages are highly polyphagous, selecting preferentially Asteraceae Fabaceae plant families, in particular some genera, e.g., Picris, Crepis, Sonchus, Bellis, Cichorium, and Medicago. Host-plant preference of nymphs varies according to the Region and through time and nymphal instar. In the monitored sites, adults peak on olive trees earlier in Apulia (i.e., during inflorescence emergence) than in Liguria (i.e., during flowering and beginning of fruit development). Principal alternative woody hosts are Quercus spp. and Pistacia spp. Knowledge concerning plant selection and ecological traits of spittlebugs in different Mediterranean olive production areas is needed to design effective and precise control strategies against X. fastidiosa vectors in olive groves, such as ground cover modifications to reduce populations of spittlebug vectors.

Beta-Cell-Specific Expression of Nicotinamide Adenine Dinucleotide Phosphate Oxidase 5 Aggravates High-Fat Diet-Induced Impairment of Islet Insulin Secretion in Mice.

Aims: Nicotinamide adenine dinucleotide phosphate oxidases (NOX-es) produce reactive oxygen species and modulate ß-cell insulin secretion. Islets of type 2 diabetic subjects present elevated expression of NOX5. Here, we sought to characterize regulation of NOX5 expression in human islets in vitro and to uncover the relevance of NOX5 in islet function in vivo using a novel mouse model expressing NOX5 in doxycycline-inducible, ß-cell-specific manner (RIP/rtTA/NOX5 mice). Results:In situ hybridization and immunohistochemistry employed on pancreatic sections demonstrated NOX5 messenger ribonucleic acid (mRNA) and protein expressions in human islets. In cultures of dispersed islets, NOX5 protein was observed in somatostatin-positive (δ) cells in basal (2.8 mM glucose) conditions. Small interfering ribonucleic acid (siRNA)-mediated knockdown of NOX5 in human islets cultured in basal glucose concentrations resulted in diminished glucose-induced insulin secretion (GIIS) in vitro. However, when islets were preincubated in high (16.7 mM) glucose media for 12 h, NOX5 appeared also in insulin-positive (ß) cells. In vivo, mice with ß-cell NOX5 expression developed aggravated impairment of GIIS compared with control mice when challenged with 14 weeks of high-fat diet. Similarly, in vitro palmitate preincubation resulted in more severe reduction of insulin release in islets of RIP/rtTA/NOX5 mice compared with their control littermates. Decreased insulin secretion was most distinct in response to theophylline stimulation, suggesting impaired cyclic adenosine monophosphate (cAMP)-mediated signaling due to increased phosphodiesterase activation. Innovation and Conclusions: Our data provide the first insight into the complex regulation and function of NOX5 in islets implying an important role for NOX5 in δ-cell-mediated intraislet crosstalk in physiological circumstances but also identifying it as an aggravating factor in ß-cell failure in diabetic conditions.

Assessment of plasma microvesicles to monitor pancreatic islet graft dysfunction: Beta cell- and leukocyte-derived microvesicles as specific features in a pilot longitudinal study.

Markers of early pancreatic islet graft dysfunction and its causes are lacking. We monitored 19 type 1 diabetes islet-transplanted patients for up to 36 months following last islet injection. Patients were categorized as Partial (PS) or complete (S) Success, or Graft Failure (F), using the ß-score as an indicator of graft function. F was the subset reference of maximum worsened graft outcome. To identify the immune, pancreatic, and liver contribution to the graft dysfunction, the cell origin and concentration of circulating microvesicles (MVs) were assessed, including MVs from insulin-secreting ß-cells typified by polysialic acid of neural cell adhesion molecule (PSA-NCAM), and data were compared with values of the ß-score. Similar ranges of PSA-NCAM+ -MVs were found in healthy volunteers and S patients, indicating minimal cell damage. In PS, a 2-fold elevation in PSA-NCAM+ -MVs preceded each ß-score drop along with a concomitant rise in insulin needs, suggesting ß-cell damage or altered function. Significant elevation of liver asialoglycoprotein receptor (ASGPR)+ -MVs, endothelial CD105+ -MVs, neutrophil CD66b+ -MVs, monocyte CD 14+ -MVs, and T4 lymphocyte CD4+ -MVs occurred before each ß-score drop, CD8+ -MVs increased only in F, and B lymphocyte CD19+ -MVs remained undetectable. In conclusion, PSA-NCAM+ -MVs are noninvasive early markers of transplant dysfunction, while ASGPR+ -MVs signal host tissue remodeling. Leukocyte MVs could identify the cause of graft dysfunction.

Insulin-producing organoids engineered from islet and amniotic epithelial cells to treat diabetes.

Maintaining long-term euglycemia after intraportal islet transplantation is hampered by the considerable islet loss in the peri-transplant period attributed to inflammation, ischemia and poor angiogenesis. Here, we show that viable and functional islet organoids can be successfully generated from dissociated islet cells (ICs) and human amniotic epithelial cells (hAECs). Incorporation of hAECs into islet organoids markedly enhances engraftment, viability and graft function in a mouse type 1 diabetes model. Our results demonstrate that the integration of hAECs into islet cell organoids has great potential in the development of cell-based therapies for type 1 diabetes. Engineering of functional mini-organs using this strategy will allow the exploration of more favorable implantation sites, and can be expanded to unlimited (stem-cell-derived or xenogeneic) sources of insulin-producing cells.

Macrophage migration inhibitory factor regulates TLR4 expression and modulates TCR/CD3-mediated activation in CD4+ T lymphocytes.

Toll-like receptor 4 (TLR4) is involved in CD4+ T lymphocyte-mediated pathologies. Here, we demonstrate that CD4+ T lymphocytes express functional TLR4 that contributes to their activation, proliferation and cytokine secretion. In addition, we demonstrate that TLR4-induced responses are mediated by macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine. We also demonstrate that MIF regulates suboptimal TCR/CD3-mediated activation of T lymphocytes. On one hand, MIF prevents excessive TCR/CD3-mediated activation of CD4+ T lymphocytes under suboptimal stimulation conditions and, on the other hand, MIF enables activated CD4+ T lymphocytes to sense their microenvironment and adapt their effector response through TLR4. Therefore, MIF appears to be a major regulator of the activation of CD4+ T lymphocytes and the intensity of their effector response. TLR4-mediated activation is thus an important process for T cell-mediated immunity.

Chronic fructose renders pancreatic ß-cells hyper-responsive to glucose-stimulated insulin secretion through extracellular ATP signaling.

Fructose is widely used as a sweetener in processed food and is also associated with metabolic disorders, such as obesity. However, the underlying cellular mechanisms remain unclear, in particular, regarding the pancreatic ß-cell. Here, we investigated the effects of chronic exposure to fructose on the function of insulinoma cells and isolated mouse and human pancreatic islets. Although fructose per se did not acutely stimulate insulin exocytosis, our data show that chronic fructose rendered rodent and human ß-cells hyper-responsive to intermediate physiological glucose concentrations. Fructose exposure reduced intracellular ATP levels without affecting mitochondrial function, induced AMP-activated protein kinase activation, and favored ATP release from the ß-cells upon acute glucose stimulation. The resulting increase in extracellular ATP, mediated by pannexin1 (Panx1) channels, activated the calcium-mobilizer P2Y purinergic receptors. Immunodetection revealed the presence of both Panx1 channels and P2Y1 receptors in ß-cells. Addition of an ectonucleotidase inhibitor or P2Y1 agonists to naïve ß-cells potentiated insulin secretion stimulated by intermediate glucose, mimicking the fructose treatment. Conversely, the P2Y1 antagonist and Panx1 inhibitor reversed the effects of fructose, as confirmed using Panx1-null islets and by the clearance of extracellular ATP by apyrase. These results reveal an important function of ATP signaling in pancreatic ß-cells mediating fructose-induced hyper-responsiveness.

Diabetes relief in mice by glucose-sensing insulin-secreting human α-cells.

Cell-identity switches, in which terminally differentiated cells are converted into different cell types when stressed, represent a widespread regenerative strategy in animals, yet they are poorly documented in mammals. In mice, some glucagon-producing pancreatic α-cells and somatostatin-producing δ-cells become insulin-expressing cells after the ablation of insulin-secreting ß-cells, thus promoting diabetes recovery. Whether human islets also display this plasticity, especially in diabetic conditions, remains unknown. Here we show that islet non-ß-cells, namely α-cells and pancreatic polypeptide (PPY)-producing γ-cells, obtained from deceased non-diabetic or diabetic human donors, can be lineage-traced and reprogrammed by the transcription factors PDX1 and MAFA to produce and secrete insulin in response to glucose. When transplanted into diabetic mice, converted human α-cells reverse diabetes and continue to produce insulin even after six months. Notably, insulin-producing α-cells maintain expression of α-cell markers, as seen by deep transcriptomic and proteomic characterization. These observations provide conceptual evidence and a molecular framework for a mechanistic understanding of in situ cell plasticity as a treatment for diabetes and other degenerative diseases.

Pest categorisation of non-EU viruses and viroids of Vitis L.

Following a request from the EU Commission, the Panel on Plant Health addressed the pest categorisation of the viruses and viroids of Vitis L. determined as being either non-EU or of undetermined standing in a previous EFSA opinion. These infectious agents belong to different genera and are heterogeneous in their biology. With the exclusion of grapevine virus 101-14.N.23.9.1/South Africa/2009 for which very limited information exists, the pest categorisation was completed for 30 viruses or viroids having acknowledged identities and available detection methods. All these viruses are efficiently transmitted by vegetative propagation techniques, with plants for planting representing the major pathway for long-distance dispersal and thus considered as the major pathway for potential entry. Depending on the virus, additional pathway(s) can also be seeds, pollen and/or vector(s). Most of the viruses categorised here are known to infect only one or few plant genera, but some of them have a wide host range, thus extending the possible entry pathways. Grapevine yellow speckle viroid 2, blueberry leaf mottle virus, grapevine Ajinashika virus, grapevine Anatolian ringspot virus, grapevine berry inner necrosis virus, grapevine deformation virus, grapevine fabavirus, grapevine red blotch virus, grapevine stunt virus, grapevine Tunisian ringspot virus, grapevine vein-clearing virus, temperate fruit decay-associated virus, peach rosette mosaic virus, tobacco ringspot virus, tomato ringspot virus meet all the criteria evaluated by EFSA to qualify as potential Union quarantine pests (QPs). With the exception of impact for the EU territory, on which the Panel was unable to conclude, blackberry virus S, grapevine geminivirus A, grapevine leafroll-associated virus 7, grapevine leafroll-associated virus 13, grapevine satellite virus, grapevine virus E, grapevine virus I, grapevine virus J, grapevine virus S, summer grape enamovirus, summer grape latent virus satisfy all the other criteria to be considered as potential Union QPs. Australian grapevine viroid, grapevine cryptic virus 1, grapevine endophyte endornavirus and wild vitis virus 1 do not meet all the criteria evaluated by EFSA to be regarded as potential Union QPs because they are not known to cause an impact on Vitis. For several viruses, especially those recently discovered, the categorisation is associated with high uncertainties mainly because of the absence of data on their biology, distribution and impact. Since this opinion addresses specifically non-EU viruses, in general these viruses do not meet the criteria assessed by EFSA to qualify as a potential Union regulated non-quarantine pests.

Pest categorisation of non-EU viruses and viroids of Prunus L.

Following a request from the EU Commission, the Panel on Plant Health addressed the pest categorisation of the viruses and viroids of Prunus L. determined as being either non-EU or of undetermined standing in a previous EFSA opinion. These infectious agents belong to different genera and are heterogeneous in their biology. With the exclusion of Ilarvirus S1 and Ilarvirus S2, for which very limited information exists, the pest categorisation was completed for 26 viruses and 1 viroid having acknowledged identities and available detection methods. All these viruses are efficiently transmitted by vegetative plant propagation techniques, with plants for planting representing the major pathway for long-distance dispersal and thus considered as the major pathway for entry. Depending on the virus, additional pathway(s) can also be Prunus seeds, pollen and/or vector(s). Most of the viruses categorised here are known to infect only one or few plant genera, but some of them have a wide host range, thus extending the possible entry pathways. Apple scar skin viroid, American plum line pattern virus, cherry mottle leaf virus, cherry rasp leaf virus, cherry rosette virus, cherry rusty mottle-associated virus, cherry twisted leaf-associated virus, peach enation virus, peach mosaic virus, peach rosette mosaic virus, tobacco ringspot virus and tomato ringspot virus meet all the criteria evaluated by EFSA to qualify as potential Union quarantine pests (QPs). With the exception of impact in the EU territory, on which the Panel was unable to conclude, apricot vein clearing virus, Asian prunus virus 1, Asian prunus virus 2, Asian prunus virus 3, Caucasus prunus virus, cherry virus B, Mume virus A, nectarine stem pitting-associated virus, nectarine virus M, peach chlorotic mottle virus, peach leaf pitting-associated virus, peach virus D, prunus virus F and prunus virus T satisfy all the other criteria to be considered as potential Union QPs. Prunus geminivirus A does not meet the criterion of having negative impact in the EU. For several viruses, especially those recently discovered, the categorisation is associated with high uncertainties mainly because of the absence of data on their biology, distribution and impact. Since this opinion addresses specifically the non-EU viruses, in general these viruses do not meet the criteria assessed by EFSA to qualify as potential Union regulated non-quarantine pests.

Electrochemically Synthesized Silver Nanoparticles Are Active Against Planktonic and Biofilm Cells of Pseudomonas aeruginosa and Other Cystic Fibrosis-Associated Bacterial Pathogens.

A novel, electrochemically synthesized, silver nanoparticles (AgNPs) formulation was evaluated in vitro against Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia, and Staphylococcus aureus strains from cystic fibrosis (CF) patients. AgNPs were particularly active against P. aeruginosa and B. cepacia planktonic cells (median MIC: 1.06 and 2.12 µg/ml, respectively) by a rapid, bactericidal and concentration-dependent effect. AgNPs showed to be particularly effective against P. aeruginosa and S. aureus biofilm causing a viability reduction ranging from 50% (1×MIC) to >99.9% (4×MIC). Electron microscopy showed that AgNPs deconstruct extracellular matrix of P. aeruginosa biofilm, and accumulate at the cell surface causing cell death secondary to membrane damage. Compared to Tobramycin, AgNPs showed comparable, or even better, activity against planktonic and biofilm P. aeruginosa cells. AgNPs at concentrations effective against B. cepacia and P. aeruginosa were not toxic to G. mellonella larvae. Our silver-based formulation might be an alternative to antibiotics in CF patients. Further in vitro and in vivo studies are warranted to confirm this therapeutic potential.

LRH-1 agonism favours an immune-islet dialogue which protects against diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is due to the selective destruction of islet beta cells by immune cells. Current therapies focused on repressing the immune attack or stimulating beta cell regeneration still have limited clinical efficacy. Therefore, it is timely to identify innovative targets to dampen the immune process, while promoting beta cell survival and function. Liver receptor homologue-1 (LRH-1) is a nuclear receptor that represses inflammation in digestive organs, and protects pancreatic islets against apoptosis. Here, we show that BL001, a small LRH-1 agonist, impedes hyperglycemia progression and the immune-dependent inflammation of pancreas in murine models of T1DM, and beta cell apoptosis in islets of type 2 diabetic patients, while increasing beta cell mass and insulin secretion. Thus, we suggest that LRH-1 agonism favors a dialogue between immune and islet cells, which could be druggable to protect against diabetes mellitus.

Variable Membrane Protein A of Flavescence Dorée Phytoplasma Binds the Midgut Perimicrovillar Membrane of Euscelidius variegatus and Promotes Adhesion to Its Epithelial Cells.

Phytoplasmas are uncultivated plant pathogens and cell wall-less bacteria and are transmitted from plant to plant by hemipteran insects. The phytoplasma's circulative propagative cycle in insects requires the crossing of the midgut and salivary glands, and primary adhesion to cells is an initial step toward the invasion process. The flavescence dorée (FD) phytoplasma possesses a set of variable membrane proteins (Vmps) exposed on its surface, and this pathogen is suspected to interact with insect cells. The results showed that VmpA is expressed by the flavescence dorée phytoplasma present in the midgut and salivary glands. Phytoplasmas cannot be cultivated at present, and no mutant can be produced to investigate the putative role of Vmps in the adhesion of phytoplasma to insect cells. To overcome this difficulty, we engineered the Spiroplasma citri mutant G/6, which lacks the ScARP adhesins, for VmpA expression and used VmpA-coated fluorescent beads to determine if VmpA acts as an adhesin in ex vivo adhesion assays and in vivo ingestion assays. VmpA specifically interacted with Euscelidiusvariegatus insect cells in culture and promoted the retention of VmpA-coated beads to the midgut of E. variegatus In this latest case, VmpA-coated fluorescent beads were localized and embedded in the perimicrovillar membrane of the insect midgut. Thus, VmpA functions as an adhesin that could be essential in the colonization of the insect by the FD phytoplasmas.IMPORTANCE Phytoplasmas infect a wide variety of plants, ranging from wild plants to cultivated species, and are transmitted by different leafhoppers, planthoppers, and psyllids. The specificity of the phytoplasma-insect vector interaction has a major impact on the phytoplasma plant host range. As entry into insect cells is an obligate process for phytoplasma transmission, the bacterial adhesion to insect cells is a key step. Thus, studying surface-exposed proteins of phytoplasma will help to identify the adhesins implicated in the specific recognition of insect vectors. In this study, it is shown that the membrane protein VmpA of the flavescence dorée (FD) phytoplasma acts as an adhesin that is able to interact with cells of Euscelidiusvariegatus, the experimental vector of the FD phytoplasma.