Aberrant Phenotype Research Articles

Metabolic Flexibility Assists Reprograming of Central and Peripheral Innate Immunity During Neurodevelopment.

Central innate immunity assists time-dependent neurodevelopment by recruiting and interacting with peripheral immune cells. Microglia are the major player of central innate immunity integrating peripheral signals arising from the circumventricular regions lacking the blood-brain barrier (BBB), via neural afferent pathways such as the vagal nerve and also by choroid plexus into the brain ventricles. Defective and/or unrestrained activation of central and peripheral immunity during embryonic development might set an aberrant connectome establishment and brain function, leading to major psychiatric disorders in postnatal stages. Molecular candidates leading to central and peripheral innate immune overactivation identified metabolic substrates and lipid species as major contributors of immunological priming, supporting the role of a metabolic flexibility node during trained immunity. Mechanistically, trained immunity is established by an epigenetic program including DNA methylation and histone acetylation, as the major molecular epigenetic signatures to set immune phenotypes. By definition, immunological training sets reprogramming of innate immune cells, enhancing or repressing immune responses towards a second challenge which potentially might contribute to neurodevelopment disorders. Notably, the innate immune training might be set during pregnancy by maternal immune activation stimuli. In this review, we integrate the most valuable scientific evidence supporting the role of metabolic cues assisting metabolic flexibility, leading to innate immune training during development and its effects on aberrant neurological phenotypes in the offspring. We also add reports supporting the role of methylation and histone acetylation signatures as a major epigenetic mechanism regulating immune training.

Aberrant Phenotypes in Acute Myeloid Leukemia and Its Relationship with Prognosis and Survival: A Systematic Review and Meta-Analysis.

Background: The aim of this review was to evaluate the influence of aberrant phenotypes in prognosis and survival in acute myeloid leukemia (AML) patients by multiparametric flow cytometry. Materials and Methods: Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, a review of PubMed, Scopus, Science Direct and Web of Science was carried out through 1998 to 2016, conducted by two reviewers independently, evaluating titles, abstracts and full-texts of the selected studies. Results: Ten studies were included on this review, in which the aberrant phenotype expression of 17 markers were detected in AML patients. From these, 11 aberrant phenotypes were associated with prognosis, which eight had shown negative impact on prognosis: CD7, CD56, CD15, CD2, CD3, CD90low, CD123high, CD117high, and three others were associated with good prognosis: CD19, CD98high and CD117+/CD15+. Meta-analysis showed that aberrant expression of CD56 as a poor prognostic marker with unfavorable outcomes is implicated in decreased overall survival in AML patients in 28 months (95% CI: 0.62 to 0.92). Conclusion: This was observed when there was association between CD56 expression and other prognostic factors, influencing on patients’ management care and treatment.

MiRNAs, lncRNAs, and circular RNAs as mediators in hypertension-related vascular smooth muscle cell dysfunction.

Hypertension is a multifactorial disorder that involves complex genetic and environmental factors. Vascular smooth muscle cells (VSMCs) are important components of blood vessels, and their dysregulation has been shown to be involved in vascular remodeling during the development of systemic hypertension and pulmonary arterial hypertension (PAH) via multiple mechanisms, such as aberrant apoptosis, phenotype conversion, proliferation, and migration of VSMCs. With increasing advances in microarrays and next-generation sequencing, nonprotein-coding RNAs (ncRNAs) have attracted much attention due to their numerous functions in health and diseases. Among ncRNAs, microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs are emerging as novel modulators in the biological behaviors of VSMCs, especially in systemic hypertension and PAH. Studies have recommended miRNAs, lncRNAs, and circular RNAs as predictive biomarkers and therapeutic targets for systemic hypertension and PAH. In this review, we summarize the current studies focusing on the roles of VSMC-derived miRNAs, lncRNAs and circular RNAs in the pathologies of systemic hypertension and PAH. MiRNAs, lncRNAs, and circular RNAs might serve as attractive targets for the prevention and treatment of VSMC dysfunction-linked systemic hypertension and PAH.

A large‐scale survey of bird plumage colour aberrations reveals a collection bias in Internet‐mined photographs

Birds with plumage colour aberrations are of interest to both the general public and scientists. However, due to their rarity in nature, information on the presence of colour aberrations is rarely found in the peer‐reviewed literature. Exploration of public observations using modern information technologies such as Internet‐based search engines could facilitate cost‐effective and rapid broad‐scale collection of data on phenotypic aberrations in animals but may also be prone to the same problems as fieldwork, including systematic collection bias. We used Google Images and also asked birdwatchers and ornithologists, via naturalists' forums and social media, to collate a unique dataset of photographs of 936 aberrantly coloured birds of 74 species from Poland. Phylogenetically informed analyses, which included species both with and without reported colour aberrations, revealed that the number of colour aberrations was higher in species with larger populations in Poland, those with larger body size, and those associated with human settlements. Colour aberrations were also more often reported for species with a wider habitat breadth and those which do not migrate over long distances. Habitat openness and diet type were not related to the number of colour aberrations across species in multivariate models. Our study emphasizes not only the power of novel sources to collect large datasets on relatively rare phenotypic aberrations in animals but also the importance of vigilance when using data mined from public sources because the observed patterns may reflect collection bias rather than the nature of the studied phenomena.

Dendritic spine abnormalities correlate with behavioral and cognitive deficits in mouse models of Lafora disease.

Lafora disease (LD) is a genetic and fatal form of neurodegenerative disorder characterized by myoclonic epilepsy and cognitive deficits. LD is caused by loss-of-function mutations in the EPM2A or the NHLRC1 gene. A major hallmark of LD is the presence of abnormal glycogen aggregates in neurons and other tissues. Functional studies on the genes have, therefore, mostly focused on glycogen metabolism. The physiological basis of cognitive deficits in LD is thus largely unexplored. Alterations in dendritic spine morphology are known in neurodevelopmental and neuropsychiatric disorders. We, therefore, analyzed the dendritic spine morphologies in pyramidal neurons of the hippocampal and Cortical layer V of the Epm2a or Nhlrc1 knockout mice brain. We found a significant increase in the density, length, and reduction in the width of the dendritic spines in Postnatal day 21 to 12-month-old LD animals. Similar observations were made in the primary cultures of neurons derived from the hippocampi of the embryonic brain, suggesting that the aberrant spine phenotype could be a developmental defect in LD. We also looked at the cognitive and behavioral deficits as a possible readout of the spine abnormalities. The LD animals exhibited hyperactivity, reduced anxiety-like behavior, and deficits in the spatial and nonspatial memory. Such abnormalities were seen in the younger (1-2 months) as well as the older (7-8 months) age groups. Taken together, our results suggest that the dendritic spine abnormalities are primary developmental defects in the LD model and these defects might underlie some of the symptoms, including cognitive deficits, in LD.

Brain activation and connectivity in anorexia nervosa and body dysmorphic disorder when viewing bodies: relationships to clinical symptoms and perception of appearance.

Anorexia nervosa (AN) and body dysmorphic disorder (BDD) are characterized by distorted perception of appearance, yet no studies have directly compared the neurobiology associated with body perception. We compared AN and BDD in brain activation and connectivity in relevant networks when viewing images of others' bodies and tested their relationships with clinical symptoms and subjective appearance evaluations. We acquired fMRI data from 64 unmedicated females (20 weight-restored AN, 23 BDD, 21 controls) during a matching task using unaltered or spatial-frequency filtered photos of others' bodies. Using general linear model and independent components analyses we compared brain activation and connectivity in visual, striatal, and parietal networks and performed univariate and partial least squares multivariate analyses to investigate relationships with clinical symptoms and appearance evaluations. AN and BDD showed partially overlapping patterns of hyperconnectivity in the dorsal visual network and hypoconnectivity in parietal network compared with controls. BDD, but not AN, demonstrated hypoactivity in dorsal visual and parietal networks compared to controls. Further, there were significant activity and connectivity differences between AN and BDD in both networks. In both groups, activity and/or connectivity were associated with symptom severity and appearance ratings of others' bodies. Thus, AN and BDD demonstrate both distinct and partially-overlapping aberrant neural phenotypes involved in body processing and visually encoding global features. Nevertheless, in each disorder, aberrant activity and connectivity show relationships to clinically relevant symptoms and subjective perception. These results have implications for understanding distinct and shared pathophysiology underlying perceptual distortions of appearance and may inform future novel treatment strategies.

MM-098: A Case Report: Acquired Pure Red Cell Aplasia Associated with Monoclonal Gammopathy of Undetermined Significance

Background: Pure Red Cell Aplasia (PRCA) is a rare disorder characterized by profound anemia and absence of erythroid precursor in the bone marrow, and it can be genetic or acquired; the acquired form can be idiopathic or secondary and one of these causes is monoclonal gammopathy (MG). In review of the literature, we found a few cases that were treated in different ways. Case Presentation: We will report a case of pure red cell aplasia associated with monoclonal gammopathy of undetermined significance; the patient admitted for severe anemia with hemoglobin 5.4 g/dl, MCV 120 fl, and white blood count (WBC) was 3750/mm3 with 67% neutrophils and a normal platelet count. The workup was done and showed a reticulocyte count of 0.03%, negative coombs (direct and indirect), negative ANA, and anti-double stranded DNA (dsDNA). Vitamin B12 was 900 pg/ml and folate 7.66 ng/ml; serology for HIV, HBV, and HCV found to be negative. TSH, LDH, liver function tests were all normal. Erythropoietin level was 11.79 IU/L. Bone marrow aspirate revealed a cellular bone marrow (60-70%) with less than 5% plasma cells expressing excess lambda light chain. Immunohistochemistry using CD138 confirmed the result and flow cytometry revealed a small population of plasma cells with aberrant phenotype. Serum protein electrophoresis showed a monoclonal gamma peak and immunofixation showed high light lambda chains. Total body MRI showed diffuse infiltrative marrow disorder. Patient was treated with Lenalidomide 25 mg daily on days 1-21 of repeated 28-day cycles and Dexamethaxone 8 mg twice weekly with excellent response. After 6 months of treatment and follow-up, patient had normal bone marrow cytology, normal hemoglobin, and normal reticulocyte count.

A Phase 2 Study of AMO-02 (Tideglusib) in Congenital and Childhood-Onset Myotonic Dystrophy Type 1 (DM1)

BackgroundGSK3β is an intracellular regulatory kinase that is dysregulated in multiple tissues in type 1 myotonic dystrophy, a rare neuromuscular disorder that manifests at any age. AMO-02 (tideglusib) inhibits GSK3β activity in preclinical models of type 1 myotonic dystrophy and promotes cellular maturation as well as normalizes aberrant molecular and behavioral phenotypes. This phase 2 study assessed the pharmacokinetics, safety and tolerability, and preliminary efficacy of AMO-02 in adolescents and adults with congenital and childhood-onset type 1 myotonic dystrophy. MethodsSixteen subjects (aged 13 to 34 years) with congenital and childhood-onset type 1 myotonic dystrophy received 12 weeks of single-blind fixed-dose oral treatment with either 400 mg (n = 8) or 1000 mg (n = 8) AMO-02 (NCT02858908). Blood samples were obtained for pharmacokinetic assessment. Safety assessments, such as laboratory tests and electrocardiograms, as well as efficacy assessments of syndromal, cognitive, and muscular functioning, were obtained. ResultsAMO-02 plasma concentrations conformed to a two-compartment model with first-order absorption and elimination, and dose-dependent increases in exposure (area under the curve) were observed. AMO-02 was generally safe and well-tolerated. No early discontinuations due to adverse events or dose adjustments of AMO-02 occurred. The majority of subjects manifested clinical improvement in their central nervous system and neuromuscular symptoms after 12 weeks of treatment compared with the placebo baseline, with a larger response noted at the 1000 mg/day dose level. AMO-02 exposure (cumulative area under the curve) was significantly correlated (P < 0.01) with change from baseline on several key efficacy assessments. ConclusionAMO-02 has favorable pharmacokinetic and clinical risk/benefit profiles meriting further study as a potential treatment for congenital and childhood-onset type 1 myotonic dystrophy.

Astrocyte regional diversity in ALS includes distinct aberrant phenotypes with common and causal pathological processes

Astrocytes are major contributors of motor neuron (MN) degeneration in amyotrophic lateral sclerosis (ALS). We investigated whether regional and cell maturation differences influence ALS astrocyte malfunction. Spinal and cortical astrocytes from SOD1G93A (mSOD1) 7-day-old mice were cultured for 5 and 13 days in vitro (DIV). Astrocyte aberrancies predominated in 13DIV cells with region specificity. 13DIV cortical mSOD1 astrocytes showed early morphological changes and a predominant reactive and inflammatory phenotype, while repressed proteins and genes were found in spinal cells. Inflammatory-associated miRNAs, e.g. miR-155/miR-21/miR-146a, were downregulated in the first and upregulated in the later ones. Interestingly, depleted miR-155/miR-21/miR-146a in small extracellular vesicles (sEVs/exosomes) was a common pathological feature. Cortical mSOD1 astrocytes induced late apoptosis and kinesin-1 downregulation in mSOD1 NSC-34 MNs, whereas spinal cells upregulated dynein, while decreased nNOS and synaptic-related genes. Both regional-distinct mSOD1 astrocytes enhanced iNOS gene expression in mSOD1 MNs. We provide information on the potential contribution of astrocytes to ALS bulbar-vs. spinal-onset pathology, local influence on neuronal dysfunction and their shared miRNA-depleted exosome trafficking. These causal and common features may have potential therapeutic implications in ALS. Future studies should clarify if astrocyte-derived sEVs are active players in ALS-related neuroinflammation and glial activation.

Decreased autophagy and fuel switching occur in a senescent hepatic cell model system.

Although aging in the liver contributes to the development of chronic liver diseases such as NAFLD and insulin resistance, little is known about the molecular and metabolic details of aging in hepatic cells. To examine these issues, we used sequential oxidative stress with hydrogen peroxide to induce premature senescence in AML12 hepatic cells. The senescent cells exhibited molecular and metabolic signatures, increased SA-βGal and γH2A.X staining, and elevated senescence and pro-inflammatory gene expression that resembled livers from aged mice. Metabolic phenotyping showed fuel switching towards glycolysis and mitochondrial glutamine oxidation as well as impaired energy production. The senescent AML12 cells also had increased mTOR signaling and decreased autophagy which likely contributed to the fuel switching from β-oxidation that occurred in normal AML12 cells. Additionally, senescence-associated secretory phenotype (SASP) proteins from conditioned media of senescent cells sensitized normal AML12 cells to palmitate-induced toxicity, a known pathological effect of hepatic aging. In summary, we have generated senescent AML12 cells which displayed the molecular hallmarks of aging and also exhibited the aberrant metabolic phenotype, mitochondrial function, and cell signaling that occur in the aged liver.

Insulin signaling and glucose metabolism in different hepatoma cell lines deviate from hepatocyte physiology toward a convergent aberrant phenotype

Hepatoma cell lines are widely used to model the hepatocyte for insulin signaling and fatty liver disease. However, a direct comparison of insulin action in primary hepatocytes and in hepatoma cell lines is needed to validate this model and to better understand liver cancer. Here we have investigated insulin signaling, gluconeogenic gene expression, glucose production, and fatty acid synthase abundance in primary hepatocytes and in HepG2, Hepa 1–6, and McARH7777 hepatoma cell lines. Differences in the electrophoretic profiles of protein extracts from human and mouse primary hepatocytes and the hepatoma cells lines are shown. Compared to primary hepatocytes, hepatoma cells showed high basal phosphorylation of AKT at Thr 308 and constitutively activated RAS-MAPK signaling, which were resistant to the dominant negative Ras mutant H-Ras17N. Hepatoma cell lines also showed defective expression of gluconeogenic enzymes, insulin unresponsive GSK phosphorylation, and marginal glucose production. Hepatoma cells also showed lower protein levels of fatty acid synthase and a largely distinct protein electrophoresis profile from hepatocytes but similar between different hepatoma lines. We conclude that hepatoma cell lines do not accurately model the hepatocyte for insulin action but may be valuable tools to investigate the proteomic changes conferring to hepatocellular carcinoma its peculiar metabolisms.

Detection of Fusion Genes to Determine Minimal Residual Disease in Leukemia Using Next-Generation Sequencing.

Measuring minimal residual disease (MRD), the persistence of leukemic cells after treatment, is important for monitoring leukemia recurrence. The current methods for monitoring MRD are flow cytometry, to assess aberrant immune phenotypes, and digital droplet PCR (ddPCR), to target genetic aberrations such as single-nucleotide variants and gene fusions. We present the performance of an RNA-based next-generation sequencing (NGS) method for MRD gene fusion detection compared with ddPCR. This method may have advantages, including the capacity to analyze different genetic aberrations and patients in 1 experiment. In particular, detection at the RNA level may be highly sensitive if the genetic aberration is highly expressed. We designed a probe-based NGS panel targeting the breakpoints of 11 fusion genes previously identified in clinical patients and 2 fusion genes present in cell lines. Blocking probes were added to prevent nonspecific enrichment. Each patient RNA sample was diluted in background RNA, depleted for rRNA and globin mRNA, converted to cDNA, and prepared for sequencing. Unique sequence reads, identified by unique molecular identifiers, were aligned directly to reference transcripts. The same patient and cell-line samples were also analyzed with ddPCR for direct comparison. Our NGS method reached a maximum sensitivity of 1 aberrant cell in 10000 cells and was mostly within a factor of 10 compared with ddPCR. Our detection limit was below the threshold of 1:1000 recommended by European Leukemia Net. Further optimizations are easy to implement and are expected to boost the sensitivity of our method to diagnostically obtained ddPCR thresholds.

Role of Isocitrate Dehydrogenase 2 on DNA Hydroxymethylation in Human Airway Smooth Muscle Cells.

Global DNA hydroxymethylation mediated by the TET (ten-eleven translocation) enzyme was induced in allergen-induced airway hyperresponsiveness in mouse lung tissues and specifically in isolated airway smooth muscle (ASM) cells. TET is an α-ketoglutarate (α-KG)-dependent enzyme, and the production of α-KG is catalyzed by IDH (isocitrate dehydrogenase). However, the role of IDH in the regulation of DNA hydroxymethylation in ASM cells is unknown. In comparison with nonasthmatic cells, asthmatic ASM cells exhibited higher TET activity and IDH2 (but not IDH-1 or IDH-3) gene expression levels. We modified the expression of IDH2 in ASM cells from humans with asthma by siRNA and examined the α-KG levels, TET activity, global DNA hydroxymethylation, cell proliferation, and expression of ASM phenotypic genes. Inhibition of IDH2 in asthmatic ASM cells decreased the α-KG levels, TET activity, and global DNA hydroxymethylation, and reversed the aberrant ASM phenotypes (including decreased cell proliferation and ASM phenotypic gene expression). Specifically, asthmatic cells transfected with siRNA against IDH2 showed decreased 5hmC (5-hydroxymethylcytosine) levels at the TGFB2 (transforming growth factor-β2) promoter determined by oxidative bisulfite sequencing. Taken together, our findings reveal that IDH2 plays an important role in the epigenetic regulation of ASM phenotypic changes in asthmatic ASM cells, suggesting that IDH2 is a potential therapeutic target for reversing the abnormal phenotypes seen in asthma.

110 RNA expression analysis in stage IVA-B cutaneous T-cell lymphoma to identify novel biomarkers of prognosis and diagnosis

Cutaneous T-cell lymphoma (CTCL) is a primary non-Hodgkin lymphoma and the most common primary cutaneous lymphoma. Due to its varied clinical and histological presentation, the accurate diagnosis of CTCL is difficult and therefore delayed; in advanced stages, CTCL is incurable and often fatal, though treatment-resistant phenotypes and survival time can be difficult to predict. There remains an unmet need for new and more effective diagnostic and prognostic indicators and novel therapies. To this end, malignant cells were isolated from the peripheral blood of 15 CTCL patients using antibody-magnetic bead or FACS sorting for CD3+CD4+ and CD7- and/or CD26- based on the known aberrant phenotype of the patient or positive selection for CD45RO+ CD4+ T cells in 3 healthy controls.

Fibronectin rescues aberrant phenotype of endothelial cells lacking either CCM1, CCM2 or CCM3.

Loss-of-function variants in CCM1/KRIT1, CCM2, and CCM3/PDCD10 are associated with autosomal dominant cerebral cavernous malformations (CCMs). CRISPR/Cas9-mediated CCM3 inactivation in human endothelial cells (ECs) has been shown to induce profound defects in cell-cell interaction as well as actin cytoskeleton organization. We here show that CCM3 inactivation impairs fibronectin expression and consequently leads to reduced fibers in the extracellular matrix. Despite the complexity and high molecular weight of fibronectin fibrils, our in vitro model allowed us to reveal that fibronectin supplementation restored aberrant spheroid formation as well as altered EC morphology, and suppressed actin stress fiber formation. Yet, fibronectin replacement neither enhanced the stability of tube-like structures nor inhibited the survival advantage of CCM3-/- ECs. Importantly, CRISPR/Cas9-mediated introduction of biallelic loss-of-function variants into either CCM1 or CCM2 demonstrated that the impaired production of a functional fibronectin matrix is a common feature of CCM1-, CCM2-, and CCM3-deficient ECs.

SHP2 mutations induce precocious gliogenesis of Noonan syndrome-derived iPSCs during neural development in vitro

BackgroundNoonan syndrome (NS) is a developmental disorder caused by mutations of Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2). Although NS patients have diverse neurological manifestations, the mechanisms underlying the involvement of SHP2 mutations in neurological dysfunction remain elusive.MethodsInduced pluripotent stem cells generated from dermal fibroblasts of three NS-patients (NS-iPSCs) differentiated to the neural cells by using two different culture systems, 2D- and 3D-cultured systems in vitro.ResultsHere we represent that SHP2 mutations cause aberrant neural development. The NS-iPSCs exhibited impaired development of EBs in which BMP and TGF-β signalings were activated. Defective early neuroectodermal development of NS-iPSCs recovered by inhibition of both signalings and further differentiated into NPCs. Intriguingly, neural cells developed from NS-NPCs exhibited abundancy of the glial cells, neurites of neuronal cells, and low electrophysiological property. Those aberrant phenotypes were also detected in NS-cerebral organoids. SHP2 inhibition in the NS-NPCs and NS-cerebral organoids ameliorated those anomalies such as biased glial differentiation and low neural activity.ConclusionOur findings demonstrate that SHP2 mutations contribute to precocious gliogenesis in NS-iPSCs during neural development in vitro.

P0125ELECTRON MICROSCOPIC DESCRIPTION OF A SENSITIVE DIAGNOSTIC PROXIMAL EPITHELIAL LYSOSOMAL LESION IN PATIENTS WITH CINAC/CKDU/MEN AND CNI NEPHROTOXICITY

Abstract Background and Aims In CINAC patients from Sri Lanka, we recently observed a sensitive constellation of proximal tubular cell findings including cellular/tubular atrophy, cell fragment shedding and, by electron microscopy (EM), the presence of an increased number of enlarged lysosomes. Here we focused on precisely defining the EM lysosomal phenotype and evaluating its presence in CINAC/MeN/CKDu cases and controls from other countries. Method Thirthy-two renal biopsies (18 Sri Lanka, 10 El Salvador, 1 India, 3 France) of patients with a diagnosis of CINAC (CKD 1-3A, 3B) were examined by electron microscopy (EM) in comparison to renal biopsies of normal kidneys at implantation, patients with calcineurin inhibitor (CNI) toxicity (n=17), proteinuric nephropathies (n=15), light chain disease (n=4), several cases on nephrotoxic drugs (lomustine, clomiphene, lithium, tenofovir, cisplatinum) and patients with reduced renal function of various causes (n=20). Results The aberrant lysosomal phenotypes can be defined as enlarged (&amp;gt;1.2µm) and dysmorphic with a light-medium electron-dense matrix containing dispersed dark electron-dense non-membrane bound round to irregular shaped “aggregates”. In addition, indicative clusters of 4-6 smaller lysosomes with the same features could be observed. No cristae or other features of mitochondria, autophagic vacuoles, lipofuscin/ceroid droplets, peroxisomes (marginal plate), lysosomal myeloid bodies (aminoglycoside nephropathy) or electron dense laminated lysosomal inclusions (Fabry disease) were observed. Patients with calcineurin inhibitor nephrotoxicity and several cases on nephrotoxic drugs (lomustine, clomiphene, lithium) and a subset of patients with light chain disease, all conditions that can either directly or indirectly be linked to calcineurin inhibition, presented the same lesions. We present an image set demonstrating the phenotypical consistency of the diagnostic lysosomal lesion versus similar features that are non-diagnostic. Conclusion A rather sensitive constellation of lysosomal lesions in renal proximal tubular cells was detected associated with CINAC/CKDu/MeN and CNI nephrotoxicity in several countries, suggesting a common new pathomechanistic paradigm where CINAC patients are experiencing a tubulotoxic mechanism similar to CNI nephrotoxicity.

P0126RENAL TRANSPLANT PATIENTS UNDER CALCINEURIN INHIBITOR THERAPY RAPIDLY ACQUIRE AN ABERRANT LYSOSOMAL LESION IN PROXIMAL TUBULAR CELLS

Abstract Background and Aims Calcineurin inhibitor therapy has changed the field of (renal) transplantation by considerably prolonging graft survival. Yet, all immunosuppressive calcineurin inhibitors are nephrotoxic that eventually contribute to complete scarring of the renal allograft. In renal biopsy analysis many histopathological features have been considered indicative of CNI nephrotoxicity, i.e. striped fibrosis, vascular hyalinosis, isometric tubular vacuolization, glomerulosclerosis, cellular infiltration and tubular atrophy, however, all are rather aspecific and can be secondary to many other causes. During the course of evaluating the specificity of a recently discovered proximal epithelial lysosomal lesion (i.e. multiple enlarged (&amp;gt;1,2µm) dysmorphic lysosomes containing dispersed electron dense non-membrane bound aggregates) in patients with Chronic Interstitial Nephropathy in Agricultural Communities (CINAC), we observed it to be present in renal transplant patients treated with cyclosporine or tacrolimus. Here, we test the hypothesis whether this lysosomal lesion is acquired during CNI therapy. Method A retrospective transmission electron microscopic analysis was performed to evaluate the presence of the typical lysosomal lesion on the following biopsies from renal transplant patients: 20 deceased donor implantation biopsies; 5 living donor implantation biopsies. For another 10 additional deceased donor renal allograft recipients, we evaluated implantation as well as protocol biopsies taken after 6 and 12 months of CNI treatment that started immediately after transplantation. Also included were 24 indication biopsies of CNI treated renal transplants. Results Of the total set of implantation biopsies (n=35), 2 (6%) were positive for the aberrant lysosomal phenotype on EM, whereas in the protocol and indication biopsies prevalence of the lesion was considerably higher ranging between 56% (protocol) and 80% (indication) of cases. Conclusion CNI therapy is associated with the fairly rapid appearance of a particular proximal tubular lysosomal phenotype observable on EM, that was not (or rarely) present at implantation. Whether this lesion is related to CNI toxicity and indicative for the outcome for the graft and/or patient survival after renal transplantation has to be investigated in a prospective trial.

Tissue Culture and Refreshment Techniques for Improvement of Transformation in Local Tetraploid and Diploid Potato with Late Blight Resistance as an Example.

Potato (Solanum tuberosum) is among the best producers of edible biomass in terms of yield per hectare and a variety of different regional cultivars are used as a staple commodity in many countries. However, this crop is attacked by several diseases, with the worst being the late blight disease caused by Phytophthora infestans. Stacking of resistance (R) genes from wild Solanum relatives are interesting prospects for the sustainable control of late blight. Therefore, we optimized methods for the efficient generation and screening of R-gene-containing transformants in tetraploid and diploid hybrid potato genotypes. Using these methods, a high transformation efficiency was achieved for the transformation of tetraploid and diploid potato lines with a triple resistance (3R) gene construct. Transformation efficiencies were improved by optimizing several factors affecting regeneration, including the quality of the starting plant material, and the composition of the plant growth regulators used during selective regeneration. A refreshment protocol was designed to alleviate in vitro related stress in stock plants, which significantly improved the growth vigor and resulted in a 4- to 10-fold increase in transformation efficiency. Furthermore, long-term exposure to exogenous Indole-3-butyric acid that is usually used for the initiation of roots in vitro, was found to cause aberrant morphological phenotypes in potato.

Frequency of Discrepancy between Bone Marrow Morphology and Immunophenotype in Acute Leukaemia

Background: Acute leukaemia (AL) are a heterogenous group of haematological malignancy characterized by uncontrolled clonal proliferation of haematopoietic progenitor cells. Objectives: The study was conducted to have a detailed understanding of immunophenotyping profile, the frequency of discrepancy between bone marrow morphology and immunophenotyping and importance of immunophenotyping in diagnosis of acute leukaemia. Methods: This prospective type of observational study was carried out with an aim to correlate the immunophenotype with bone&#x0D; marrow morphology and to see the discrepancy between this two in acute leukaemia. A total of 38 untreated acute leukemia patients attending in the Department of Haematology, Bangabandhu Sheikh Mujib Medical University, Dhaka, during the period from October 2016 to September 2017were included in this study. At first the morphological diagnosis was done. Then the immunophenotypic profile was compared. Result: Around eighty-two cases of acute leukaemia did find similarity with immunophenotyping and remaining 18.4% shows discrepancy. Diagnosis in this 18.4% changes after immunophenotyping. Aberrant phenotypes were detected in 20 (52.6%) samples among them 13 (34.21%) cases were AML, 3 (7.8%) cases were B-ALL and 4 (10.52%) cases were T-ALL. Significant relation was not found between aberrant marker and FAB subtypes. Conclusion: In acute leukaemia morphological appearance of bone marrow does not always match with immunophenotyping. It is therefore imperative and absolutely essential to ascertain the lineage of leukaemia by immunophenotyping before starting treatment.