The Potential of JAG Ligands as Therapeutic Targets and Predictive Biomarkers in Multiple Myeloma.

The NOTCH ligands JAG1 and JAG2 have been correlated in vitro with multiple myeloma (MM) cell proliferation, drug resistance, self-renewal and a pathological crosstalk with the tumor microenvironment resulting in angiogenesis and osteoclastogenesis. These findings suggest that a therapeutic approach targeting JAG ligands might be helpful for the care of MM patients and lead us to explore the role of JAG1 and JAG2 in a MM in vivo model and primary patient samples. JAG1 and JAG2 protein expression represents a common feature in MM cell lines; therefore, we assessed their function through JAG1/2 conditional silencing in a MM xenograft model. We observed that JAG1 and JAG2 showed potential as therapeutic targets in MM, as their silencing resulted in a reduction in the tumor burden. Moreover, JAG1 and JAG2 protein expression in MM patients was positively correlated with the presence of MM cells in patients' bone marrow biopsies. Finally, taking advantage of the Multiple Myeloma Research Foundation (MMRF) CoMMpass global dataset, we showed that JAG2 gene expression level was a predictive biomarker associated with patients' overall survival and progression-free survival, independently from other main molecular or clinical features. Overall, these results strengthened the rationale for the development of a JAG1/2-tailored approach and the use of JAG2 as a predictive biomarker in MM.

Therapeutic Effect of an Antibody-Derived Peptide in a Galleria mellonella Model of Systemic Candidiasis.

The synthetic peptide T11F (TCRVDHRGLTF), with sequence identical to a fragment of the constant region of human IgM, and most of its alanine-substituted derivatives proved to possess a significant candidacidal activity in vitro. In this study, the therapeutic efficacy of T11F, D5A, the derivative most active in vitro, and F11A, characterized by a different conformation, was investigated in Galleria mellonella larvae infected with Candida albicans. A single injection of F11A and D5A derivatives, in contrast with T11F, led to a significant increase in survival of larvae injected with a lethal inoculum of C. albicans cells, in comparison with infected animals treated with saline. Peptide modulation of host immunity upon C. albicans infection was determined by hemocyte analysis and larval histology, highlighting a different immune stimulation by the studied peptides. F11A, particularly, was the most active in eliciting nodule formation, melanization and fat body activation, leading to a better control of yeast infection. Overall, the obtained data suggest a double role for F11A, able to simultaneously target the fungus and the host immune system, resulting in a more efficient pathogen clearance.

Candida isolates causing candidemia show different degrees of virulence in Galleria mellonella.

We aim to assess intra- and interspecies differences in the virulence of Candida spp. strains causing candidemia using the invertebrate Galleria mellonella model. We studied 739 Candida spp. isolates (C. albicans [n = 373], C. parapsilosis [n = 203], C. glabrata [n = 92], C. tropicalis [n = 53], and C. krusei [n = 18]) collected from patients with candidemia admitted to Gregorio Marañon Hospital (Madrid, Spain). Species-specific infecting inocula (yeast cells/larva) were adjusted (5 × 105 [C. albicans, and C. tropicalis], 2 × 106-5 × 106 [C. parapsilosis, C. glabrata, and C. krusei]) and used to infect 10 larvae per isolate; percentage of survival and median survival per isolate were calculated. According to the interquartile range of the median survival, isolates with a median survival under P25 were classified as of high-virulence and isolates with a median survival over P75 as of low virulence. The median survival of larvae infected with different species was variable: C. albicans (n = 2 days, IQR <1-3 days), C. tropicalis (n = 2 days, IQR 1.5-4 days), C. parapsilosis (n = 2 days, IQR 2-3.5 days), C. glabrata (n = 3 days, IQR 2-3 days), and C. krusei (n = 7 days, 6.5->8 days) (P < .001). Differences in virulence among species were validated by histological examination (day +1 post-infection) in the larvae infected by the isolates of each virulence category and species. Virulence-related gene expression in C. albicans isolates did not reach statistical significance. We report species-specific virulence patterns of Candida spp. and show that isolates within a given species have different degrees of virulence in the animal model.

EPIRETINAL FIBROSIS REMOVAL IN AN ARGUS II-IMPLANTED EYE: Histological Characteristics and Functional Results.

PURPOSE: 1) To investigate morphologic and histochemical characteristics of an epiretinal fibrosis removed in an Argus II-implanted eye; 2) to evaluate the Argus II function before and after the fibrosis removal, and 3) to compare morphologic and functional data. METHODS: Fibrosis, which developed between the Argus II prosthesis and the retina two years after implant, was surgically removed. Its morphologic and histochemical characteristics were evaluated both in light and transmission electron microscopy, with special stains and immunohistochemistry. The Argus II function was evaluated during the follow-up before surgical removal and 1 month later. RESULTS: Fibrosis was successfully removed. It was composed of a fibrotic tissue with spindle cells arranged in nodular aggregates with a symmetric distribution, mixed with an inflammatory infiltrate. Extra- and intracellular, irregular, small iron particles were found and confirmed ultrastructural characterization with degenerative cellular changes. The repositioned Argus II restored, and its function was partially nearly to normal values 1 month after surgery. CONCLUSION: Fibrosis can develop between the Argus II and the retina with increasing reduced function. Morphologic characteristics of the removed fibrosis suggested a pathogenesis based on an inflammatory process involved in a foreign body reaction with progressing connective tissue deposition leading to sclerosis. Adequate clinical follow-up is critical to successful removal of the fibrosis with reactivation of the Argus II function.

OATPB1/B3 and MRP3 expression in hepatocellular adenoma predicts Gd-EOB-DTPA uptake and correlates with risk of malignancy.

BACKGROUND AND AIMS: Hepatobiliary phase (HBP) Gd-EOB-DTPA-enhanced magnetic resonance imaging (MRI) has increased the accuracy in differentiating focal nodular hyperplasia (FNH) and hepatocellular adenoma (HCA). However, the ability of this technique to distinguish HCA subtypes remains controversial. The aim of this study was to investigate the expression of hepatocyte transporters (OATPB1/B3, MRP2, MRP3) in HCA subtypes, hence to understand their MRI signal intensity on HBP Gd-EOB-DTPA-enhanced MRI. METHODS: By means of immunohistochemistry (IHC), we scored the expression of OATPB1/B3, MRP2 and MRP3, in resected specimens of FNH (n = 40), subtyped HCA (n = 58) and HCA with focal malignant transformation (HCA-HCC, n = 4). Results were validated on a supplementary set of FNH (n = 6), subtyped HCA (n = 17) and HCA-HCC (n = 1) with Gd-EOB-DTPA MR images. RESULTS: All FNH showed a preserved expression of hepatocytes transporters. Beta-catenin-activated HCA (at highest risk of malignant transformation) and HCA-HCC were characterized by preserved/increased OATPB1/B3 expression (predictor of hyperintensity on HBP), as opposed to other HCA subtypes (P < 0.01) that mostly showed OATPB1/B3 absence (predictor of hypointensity on HBP). HCA-HCC showed an additional MRP3 overexpressed profile (P < 0.01). On HBP Gd-EOB-DTPA-enhanced MRI, FNH and HCA signal intensity reflected the profile predicted by their specific OATPB1/B3 tissue expression. The hyperintense vs hypointense HBP signal criterion was able to distinguish all higher risk HCA and HCA-HCC (100% accuracy). CONCLUSIONS: OATPB1/B3 and MRP3 IHC and signal intensity on HBP Gd-EOB-DTPA-enhanced MRI can help to stratify HCA according to their risk of malignant transformation.

Therapeutic Effect of Iron Citrate in Blocking Calcium Deposition in High Pi-Calcified VSMC: Role of Autophagy and Apoptosis.

In chronic kidney disease (CKD), the first cause of mortality is cardiovascular disease induced mainly by vascular calcification (VC). Recently, iron-based phosphate binders have been proposed in advanced CKD to treat hyperphosphatemia. We studied the effect of iron citrate (iron) on the progression of calcification in high-phosphate (Pi) calcified VSMC. Iron arrested further calcification when added on days 7-15 in the presence of high Pi (1.30 ± 0.03 vs 0.61 ± 0.02; OD/mg protein; day 15; Pi vs Pi + Fe, p < 0.01). We next investigated apoptosis and autophagy. Adding iron to high-Pi-treated VSMC, on days 7-11, decreased apoptotic cell number (17.3 ± 2.6 vs 11.6 ± 1.6; Annexin V; % positive cells; day 11; Pi vs Pi + Fe; p < 0.05). The result was confirmed thorough analysis of apoptotic nuclei both in VSMCs and aortic rings treated on days 7-15 (3.8 ± 0.2 vs 2.3 ± 0.3 and 4.0 ± 0.3 vs 2.2 ± 0.2; apoptotic nuclei; arbitrary score; day 15; Pi vs Pi + Fe; VSMCs and aortic rings; p < 0.05). Studying the prosurvival axis GAS6/AXL, we found that iron treatment on days 9-14 counteracted protein high-Pi-stimulated down-regulation and induced its de novo synthesis. Moreover, iron added on days 9-15 potentiated autophagy, as detected by an increased number of autophagosomes with damaged mitochondria and an increase in autophagic flux. Highlighting the effect of iron on apoptosis, we demonstrated its action in blocking the H2O2-induced increase in calcification added both before high Pi treatment and when the calcification was already exacerbated. In conclusion, we demonstrate that iron arrests further high Pi-induced calcium deposition through an anti-apoptotic action and the induction of autophagy on established calcified VSMC.

Integrin ανß5 in vitro inhibition limits pro-fibrotic response in cardiac fibroblasts of spontaneously hypertensive rats.

BACKGROUND: To date the TGF-ß1 activation mediated by integrin ανß5 during fibrosis is well-known. This process has been shown also in the heart, where cardiac fibroblasts (CF) differentiate into α-smooth muscle actin (α-SMA)-positive myofibroblasts (MyoFB). Here, we studied the effects on CF, isolated by spontaneously hypertensive rats (SHR), of integrin ανß5 inhibition in MyoFB differentiation. METHODS: Staining and immunohistochemistry were performed on rat cardiac tissue. CF were isolated by enzymatic digestion from SHR (SHR-CF) and normotensive WKY (WKY-CF) rat hearts and then treated for in vitro evaluation. RESULTS: SHR heart tissues revealed a higher TGF-ß1 expression vs. WKY samples. SHR-CF showed an enhanced SMAD2/3 activation and an up-regulated expression of α-SMA, a typical MyoFB marker, especially after TGF-ß1 treatment. Immunostaining on cardiac tissues revealed a higher expression of integrin ανß5 in SHR vs. WKY rat hearts. In vitro results confirmed the up-regulation of integrin ανß5 expression in SHR-CF at basal condition and after TGF-ß1 treatment, in comparison with WKY-CF. Inhibition of integrin ανß5 by cilengitide treatment led a decreased expression of ανß5, collagen I, and α-SMA in SHR-CF vs. WKY-CF, resulting in a diminished differentiation of CF into MyoFB. Taking together, results suggested that SHR-CF are more susceptible to TGF-ß1, showing an up-regulated activation of SMAD2/3 signaling, and an increased ανß5, α-SMA, and collagen I expression. Hypertension stimulus promoted an up-regulation of integrin ανß5 on SHR cardiac tissue and its in vitro inhibition reverted pro-fibrotic events of SHR-CF. CONCLUSION: Inhibition of integrin ανß5 exerted by cilengitide strongly diminished SHR-CF differentiation into detrimental MyoFB. So, integrin ανß5 might be considered a novel therapeutic target and cilengitide an effective pharmacological tool to limit the progression of hypertension-induced cardiac fibrosis.

Transglutaminase 2 Mediates the Cytotoxicity of Resveratrol in a Human Cholangiocarcinoma and Gallbladder Cancer Cell Lines.

Resveratrol is a polyphenolic compound extracted from plants and is also a constituent of red wine. Our aim was to evaluate if the cytotoxic effect of resveratrol (RES) on cholangiocarcinoma (CC) and gallbladder cancer (GBC) cell lines could be abolished by TG2 inhibition. Human CC and GBC cell lines (SK-ChA-1 and MZ-ChA-1), grown in a three-dimensional cell culture system (MCTS, multicellular tumor spheroids), were treated for 72 h with RES (32, 64 µM) alone or combined with different TG2 inhibitors (Cystamine, B003, T101). We investigated: cells viability; cell morphology with light microscopy (LM) and transmission electron microscopy (TEM); immunoreactivity with immunohistochemistry; Q-Banding karyotype analysis; TG2 activity; Western blotting. RES treatment induced a significant inhibition of cell growth, ranging from 24% to 76% in both cell lines. The inhibitors successfully reduced TG2 activity without any variation of protein quantity as demonstrated by immunohistochemistry and Western blot. TG2 inhibition resulted in cell growth normalization. In addition, morphologic analysis by light and transmission electron microscopy confirmed the cytotoxic effect of RES and its reduction consequent to TG2 inhibition. Our data demonstrated a connection between the cytotoxic effect of RES in SK-ChA-1 and MZ-ChA-1 and TG2 activity.

Inhibition of ceramide de novo synthesis by myriocin produces the double effect of reducing pathological inflammation and exerting antifungal activity against A. fumigatus airways infection.

BACKGROUND: Fungal infections develop in pulmonary chronic inflammatory diseases such as asthma, Chronic Obstructive Pulmonary Disease (COPD) and Cystic Fibrosis (CF). The available antifungal drugs may fail to eradicate fungal pathogens, that can invade the lungs and vessels and spread by systemic circulation taking advantage of defective lung immunity. An increased rate of sphingolipid de novo synthesis, leading to ceramide accumulation, was demonstrated in CF and COPD inflamed lungs. The inhibitor of sphingolipid synthesis myriocin reduces inflammation and ameliorates the response against bacterial airway infection in CF mice. Myriocin also inhibits sphingolipid synthesis in fungi and exerts a powerful fungistatic effect. METHODS: We treated Aspergillus fumigatus infected airway epithelial cells with myriocin and we administered myriocin-loaded nanocarriers to A. fumigatus infected mice lung. RESULTS: We demonstrate here that de novo synthesized ceramide mediates the inflammatory response induced by A. fumigatus infection in airway epithelia. CF epithelial cells are chronically inflamed and defective in killing internalized conidia. Myriocin treatment reduced ceramide increase and inflammatory mediator release whereas it upregulated HO1 and NOD2, allowing the recovery of a functional killing of conidia in these cells. Myriocin-loaded nanocarriers, intratracheally administered to mice, significantly reduced both the inflammatory response induced by A. fumigatus pulmonary challenge and fungal lung invasion. CONCLUSIONS: We conclude that inhibition of sphingolipid synthesis can be envisaged as a dual anti-inflammatory and anti-fungal therapy in patients suffering from chronic lung inflammation with compromised immunity. GENERAL SIGNIFICANCE: Myriocin represents a powerful agent for inflammatory diseases and fungal infection.

Unexpected distribution of CA19.9 and other type 1 chain Lewis antigens in normal and cancer tissues of colon and pancreas: Importance of the detection method and role of glycosyltransferase regulation.

BACKGROUND: CA19.9 antigen has been assumed as an abundant product of cancer cells, due to the reactivity found by immunohistochemical staining of cancer tissues with anti-CA19.9 antibody. METHODS: Expression and biosynthesis of type 1 chain Lewis antigens in the colon and the pancreas were studied by immunodetection in tissue sections and lysates, quantification of glycosyltransferase transcripts, bisulfite sequencing, and chromatin immunoprecipitation assays. RESULTS: CA19.9 was poorly detectable in normal colon mucosa and almost undetectable in colon cancer, while it was easily detected in the pancreatic ducts, together with Lewis b antigen, under both normal and cancer conditions. B3GALT5 transcripts were down-regulated in colon cancer, while they remained expressed in pancreatic cancer. Even ST3GAL3 transcript appeared well expressed in the pancreas but poorly in the colon, irrespective of normal or cancer conditions. CpG islands flanking B3GALT5 native promoter presented an extremely low degree of methylation in pancreatic cancer with respect to colon cancer. In a DNA region about 1kb away from the B3GALT5 retroviral promoter, a stretch of CG dinucleotides presented a methylation pattern potentially associated with transcription. Such a DNA region and the transcription factor binding site provided overlapping results by chromatin immunoprecipitation assays, corroborating the hypothesis. CONCLUSIONS: CA19.9 appears as a physiological product whose synthesis strongly depends on the tissue specific and epigenetically-regulated expression of B3GALT5 and ST3GAL3. GENERAL SIGNIFICANCE: CA19.9 and other Lewis antigens acquire tumor marker properties in the pancreas due to mechanisms giving rise to reabsorption into vessels and elevation in circulating levels.

Osteonectin (SPARC) Expression in Vascular Calcification: In Vitro and Ex Vivo Studies.

Osteonectin, also termed SPARC, is a noncollagenous protein of bone matrix. Since there are controversial results regarding its role during the process of vascular calcification, we investigated osteonectin expression in our in vitro calcification model. Rat vascular smooth muscle cells (VSMCs) were challenged with high phosphate (5 mmol/L Pi) and analyzed quantifying calcium levels, through immunohistochemical studies, and studying gene expression. We detected a peak of osteonectin expression at day 7 in cell treated with high phosphate. The time course of calcium deposition, reflected the expression of osteonectin, resulting extensively present at day 7. On the contrary, the expression of the mitotic marker Ki-67 had a peak at day 4, showing no correlation between osteonectin and cell proliferation. Moreover, 7 days was the time point in which Cbfα1/RUNX-2 had its maximal expression. Furthermore, ascorbic acid increased osteonectin expression, supporting a procalcifying role for this protein. Next we decided to study osteonectin expression ex vivo in fetal, adult not calcified, and adult calcific vessels. Immunohistochemical studies demonstrated a spread and strong reactivity in VSMCs of a 20-week fetus, confirming that osteonectin may have a potential role in regulation of mitosis and in cell differentiation. In adult not calcified arteries, osteonectin was constitutively expressed and its levels increased in atherosclerotic and in calcified plaques, where it could have a regulatory role in the calcification process. Our in vitro and ex vivo data show osteonectin expression during the calcification process and suggest its potential role as procalcifying factor.

Cell death and cell proliferation in human spina bifida.

BACKGROUND: Spina bifida is a multifactorial congenital malformation of the central nervous system. The aim of this study was to ascertain the relevance of cell death/proliferation balance in human spina bifida and to assess autophagy distribution and levels during embryo-fetal development in neural tissue. METHODS: Five human cases with myelomeningocoele were compared with 10 healthy human controls and LC3 protein expression was also analyzed in mouse embryos. Cell death was evaluated using TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling) assay; cell proliferation was studied by counting Ki67-positive cells, and autophagy was assessed by observing the presence of LC3 punctuate dots. RESULTS: Comparing human cases and controls (13 to 21 weeks of gestation), we observed a significant increase in TUNEL-positive cells in human spina bifida associated with a significantly decreased proliferation rate, indicating an alteration of the physiological cell rate homeostasis. LC3 distribution was found to be spatiotemporally regulated in both human and murine embryo-fetuses: in early pregnancy a diffuse and ubiquitous LC3 signal was detected. After neural tube closure, an intense LC3-positive signal, normally associated to extra energy requirement, was confined to the Lissauer's tract, the dorsolateral spinal zone containing centrally projecting axons from dorsal root ganglia, at any medullar levels. LC3 signal disappeared from 12 weeks of gestation. CONCLUSION: In conclusion, this study confirms the fundamental role of cell death/proliferation balance during central nervous system development and reports the changing expression of LC3 protein in mouse and human neural tube. Birth Defects Research (Part A) 106:104-113, 2016. © 2015 Wiley Periodicals, Inc.

Acetylcholine Protects against Candida albicans Infection by Inhibiting Biofilm Formation and Promoting Hemocyte Function in a Galleria mellonella Infection Model.

Both neuronal acetylcholine and nonneuronal acetylcholine have been demonstrated to modulate inflammatory responses. Studies investigating the role of acetylcholine in the pathogenesis of bacterial infections have revealed contradictory findings with regard to disease outcome. At present, the role of acetylcholine in the pathogenesis of fungal infections is unknown. Therefore, the aim of this study was to determine whether acetylcholine plays a role in fungal biofilm formation and the pathogenesis of Candida albicans infection. The effect of acetylcholine on C. albicans biofilm formation and metabolism in vitro was assessed using a crystal violet assay and phenotypic microarray analysis. Its effect on the outcome of a C. albicans infection, fungal burden, and biofilm formation were investigated in vivo using a Galleria mellonella infection model. In addition, its effect on modulation of host immunity to C. albicans infection was also determined in vivo using hemocyte counts, cytospin analysis, larval histology, lysozyme assays, hemolytic assays, and real-time PCR. Acetylcholine was shown to have the ability to inhibit C. albicans biofilm formation in vitro and in vivo. In addition, acetylcholine protected G. mellonella larvae from C. albicans infection mortality. The in vivo protection occurred through acetylcholine enhancing the function of hemocytes while at the same time inhibiting C. albicans biofilm formation. Furthermore, acetylcholine also inhibited inflammation-induced damage to internal organs. This is the first demonstration of a role for acetylcholine in protection against fungal infections, in addition to being the first report that this molecule can inhibit C. albicans biofilm formation. Therefore, acetylcholine has the capacity to modulate complex host-fungal interactions and plays a role in dictating the pathogenesis of fungal infections.

A new in vitro model to delay high phosphate-induced vascular calcification progression.

An increasing amount of patients affected by advanced chronic kidney disease suffer from vascular calcification (VC) that associates with cardiovascular morbidity and mortality. In this study, we created a new experimental in vitro model, trying to better elucidate high phosphate (Pi)-induced VC pathogenic mechanisms. Rat aortic vascular smooth muscle cells (VSMCs) were challenged for 7-10 days with high Pi with a repeated and short suspensions of high Pi treatment (intermittent suspension, IS) that was able to induce a significant inhibition of high Pi calcification, maximal at 5 h. Interestingly, the delay in calcification is a consequence of either the absence of free Pi or calcium-phosphate crystals being comparable to the total effect obtained during the 5 h-IS. The protective effect of IS was mediated by the reduction of apoptosis as demonstrated by the action of 20 µmol/L Z-VAD-FMK and by the preservation of the pro-survival receptor Axl expression. Furthermore, autophagy, during IS, was potentiated by increasing the autophagic flux, evaluated by LC3IIB western, while treating VSMCs with 1 mmol/L valproic acid did not affect VC. Finally, IS prevented VSMC osteoblastic differentiation by preserving smooth muscle lineage markers expression. Our data support the hypothesis that to delay significantly VC is necessary and sufficient the IS of high Pi challenge. The IS was able to prevent significantly apoptosis, to induce a potentiation in autophagy, and to prevent osteoblastic differentiation by preserving SM lineage markers.

Survivin family proteins as novel molecular determinants of doxorubicin resistance in organotypic human breast tumors.

INTRODUCTION: The molecular determinants of breast cancer resistance to first-line anthracycline-containing chemotherapy are unknown. METHODS: We examined the response to doxorubicin of organotypic cultures of primary human breast tumors ex vivo with respect to cell proliferation, DNA damage and modulation of apoptosis. Samples were analyzed for genome-wide modulation of cell death pathways, differential activation of p53, and the role of survivin family molecules in drug resistance. Rational drug combination regimens were explored by high-throughput screening, and validated in model breast cancer cell types. RESULTS: Doxorubicin treatment segregated organotypic human breast tumors into distinct Responder or Non Responder groups, characterized by differential proliferative index, stabilization of p53, and induction of apoptosis. Conversely, tumor histotype, hormone receptor or human epidermal growth factor receptor-2 (HER2) status did not influence chemotherapy sensitivity. Global analysis of cell death pathways identified survivin and its alternatively spliced form, survivin-ΔEx3 as uniquely overexpressed in Non Responder breast tumors. Forced expression of survivin-ΔEx3 preserved cell viability and prevented doxorubicin-induced apoptosis in breast cancer cell types. High-throughput pharmacologic targeting of survivin family proteins with a small-molecule survivin suppressant currently in the clinic (YM155) selectively potentiated the effect of doxorubicin, but not other chemotherapeutics in breast cancer cell types, and induced tumor cell apoptosis. CONCLUSIONS: Survivin family proteins are novel effectors of doxorubicin resistance in chemotherapy-naive breast cancer. The incorporation of survivin antagonist(s) in anthracycline-containing regimens may have improved clinical activity in these patients.

Biofilms formed by Candida albicans bloodstream isolates display phenotypic and transcriptional heterogeneity that are associated with resistance and pathogenicity.

BACKGROUND: Candida albicans infections have become increasingly recognised as being biofilm related. Recent studies have shown that there is a relationship between biofilm formation and poor clinical outcomes in patients infected with biofilm proficient strains. Here we have investigated a panel of clinical isolates in an attempt to evaluate their phenotypic and transcriptional properties in an attempt to differentiate and define levels of biofilm formation. RESULTS: Biofilm formation was shown to be heterogeneous; with isolates being defined as either high or low biofilm formers (LBF and HBF) based on different biomass quantification. These categories could also be differentiated using a cell surface hydrophobicity assay with 24 h biofilms. HBF isolates were more resistance to amphotericin B (AMB) treatment than LBF, but not voriconazole (VRZ). In a Galleria mellonella model of infection HBF mortality was significantly increased in comparison to LBF. Histological analysis of the HBF showed hyphal elements intertwined indicative of the biofilm phenotype. Transcriptional analysis of 23 genes implicated in biofilm formation showed no significant differential expression profiles between LBF and HBF, except for Cdr1 at 4 and 24 h. Cluster analysis showed similar patterns of expression for different functional classes of genes, though correlation analysis of the 4 h biofilms with overall biomass at 24 h showed that 7 genes were correlated with high levels of biofilm, including Als3, Eap1, Cph1, Sap5, Plb1, Cdr1 and Zap1. CONCLUSIONS: Our findings show that biofilm formation is variable amongst C. albicans isolates, and categorising isolates depending on this can be used to predict how pathogenic the isolate will behave clinically. We have shown that looking at individual genes in less informative than looking at multiple genes when trying to categorise isolates at LBF or HBF. These findings are important when developing biofilm-specific diagnostics as these could be used to predict how best to treat patients infected with C. albicans. Further studies are required to evaluate this clinically.

Primary HIV infection features colonic damage and neutrophil inflammation yet containment of microbial translocation.

INTRODUCTION: Impairment of the gastrointestinal barrier leads to microbial translocation and peripheral immune activation, which are linked to disease progression. Data in the setting of primary HIV/SIV infection suggest that gut barrier damage is one of the first events of the pathogenic cascade, preceding mucosal immune dysfunction and microbial translocation. We assessed gut structure and immunity as well as microbial translocation in acutely and chronically-infected, combination antiretroviral therapy (cART)-naive individuals. METHODS: Fifteen people with primary HIV infection (P-HIV) and 13 with chronic HIV infection (C-HIV) c-ART-naive participants were cross-sectionally studied. Gut biopsies were analysed in terms of gut reservoirs (total, integrated and unintegrated HIV DNA); tight junction proteins (E-cadherin, Zonula Occludens-1), CD4 + expression, neutrophil myeloperoxidase (histochemical staining); collagen deposition (Masson staining). Flow cytometry was used to assess γδ T-cell frequency (CD3 + panγδ+Vδ1+/Vδ2+). In plasma, we measured microbial translocation (LPS, sCD14, EndoCAb) and gut barrier function (I-FABP) markers (ELISA). RESULTS: P-HIV displayed significantly higher tissue HIV DNA, yet neutrophil infiltration and collagen deposition in the gut were similar in the two groups. In contrast, microbial translocation markers were significantly lower in P-HIV compared with C-HIV. A trend to higher mucosal E-cadherin, and gut γδ T-cells was also observed in P-HIV. CONCLUSION: Early HIV infection features higher HIV DNA in the gut, yet comparable mucosal alterations to those observed in chronic infection. In contrast, microbial translocation is contained in primary HIV infection, likely because of a partial preservation of E-cadherin and mucosal immune subsets, namely γδ T-cells.

Canine glioblastoma-derived extracellular vesicles as precise carriers for glioblastoma imaging: Targeting across the blood-brain barrier.

The treatment of glioblastoma (GBM) faces significant challenges due to the difficulty of delivering drugs through the blood-brain barrier (BBB). Extracellular vesicles (EVs) have emerged as potential carriers for targeted drug delivery to brain tumors. However, their use and distribution in the presence of an intact BBB and their ability to target GBM tissue are still under investigation. This study explored the use of EVs for GBM targeting across the BBB. Canine plasma EVs from healthy dogs and dogs with glioma were isolated, characterized, and loaded with diagnostic agents. Biodistribution studies were conducted in healthy murine models and a novel intranasal model that preserved BBB integrity while initiating early-stage GBM growth. This model assessed EVs' potential for delivering the contrast agent gadoteric acid to intracranial tumors. Imaging techniques, such as bioluminescence and MRI, confirmed EVs' targeting and delivery capabilities thus revealing a selective accumulation of canine glioma-derived EVs in brain tissue under physiological conditions. In the model of brain tumor, MRI experiments demonstrated the ability of EVs to accumulate gadoteric acid within GBM to enhance contrast of the tumoral mass, even when BBB integrity is maintained. This study underscores the potential of EVs derived from glioma for the targeted delivery of drugs to glioblastoma. EVs from dogs with glioma showed capacity to traverse the BBB and selectively accumulate within the brain tumor. Overall, this research represents a foundation for the application of autologous EVs to precision glioblastoma treatment, addressing the challenge of BBB penetration and targeting specificity in brain cancer therapy.

Lanthanum prevents high phosphate-induced vascular calcification by preserving vascular smooth muscle lineage markers.

Vascular calcification (VC) represents a major cardiovascular risk factor in chronic kidney disease patients. High phosphate (Pi) levels are strongly associated with VC in this population. Therefore, Pi binders are commonly used to control high Pi levels. The aim of this work was to study the mechanism of action of lanthanum chloride (LaCl3) on the progression of Pi-induced VC through its direct effect on vascular smooth muscle cells (VSMCs) in vitro. High Pi induced VSCM Ca deposition. We evaluated the action of LaCl3, compared to gadolinium chloride (GdCl3), and found different effects on the modulation of VSMC lineage markers, such as α-actin and SM22α. In fact, only LaCl3 preserved the expression of both VSMC lineage markers compared to high Pi-treated cells. Interestingly, both LaCl3 and GdCl3 reduced the high Pi-induced elevations of bone morphogenic protein 2 mRNA expression, with no reduction of the high core binding factor-alpha 1 mRNA levels observed in calcified VSMCs. Furthermore, we also found that only LaCl3 completely prevented the matrix GLA protein mRNA levels and osteonectin protein expression elevations induced by high Pi compared to GdCl3. Finally, LaCl3, in contrast to GdCl3, prevented the high Pi-induced downregulation of Axl, a membrane tyrosine kinase receptor involved in apoptosis. Thus, our results suggest that LaCl3 prevents VC by preserving VSMC lineage markers and by decreasing high Pi-induced osteoblastic differentiation.

Aberrant overexpression of the cell polarity module scribble in human cancer.

Human Scribble (Scrib) is an evolutionary-conserved cell polarity protein, but its potential role in human cancer is controversial. Herein, we show that Scrib is nearly universally overexpressed in cultured tumor cell lines and genetically disparate cancer patient series compared with matched normal tissues in vivo. Instead of a membrane association seen in normal epithelia, tumor-associated Scrib is mislocalized and found predominantly in the cytosol. Small-interfering RNA silencing of Scrib in model lung adenocarcinoma A549 cells inhibited cell migration in wound-healing assays, suppressed tumor cell invasion across Matrigel-coated inserts, and down-regulated the expression of cell motility markers and mediators of epithelial-mesenchymal transition. These data uncover a previously unrecognized exploitation of Scrib for aberrant tumor cell motility and invasion, thus potentially contributing to disease progression in humans.