Unraveling the molecular underpinnings of Philadelphia-negative myeloproliferative neoplasms: implications for future therapeutic strategies

The present thesis encompasses the two researches projects I conducted during my PhD program in Molecular Biology and Pathology. The common thread is represented by the analysis of the signaling pathways implicated in the pathophysiology of the two most aggressive Philadelphia-negative myeloproliferative neoplasms, namely, atypical chronic myeloid leukemia (aCML) and primary myelofibrosis (PMF). In the last decade, since the description of the JAK2V617F mutation in 2005, the field of the molecular characterization of Philadelphia-negative myeloproliferative neoplasms has experienced an astonishing implementation that led to the discovery of 16 new mutations involving signal transduction, epigenetic modifiers, cell cycle regulators. Nevertheless, their pathogenetic relevance and whether they could represent good “druggable” candidates have to be proved yet. In the first section I provide the first report of the signaling cascade down-stream the rare cytogenetic lesion t(8;9)(p22;p24)/PCM1-JAK2 associated with aCML, finding that it selectively activates the ERK1/2 signaling without affecting JAK/STAT phosphorylation. In the second part, I investigated the implication of the ε isoform of novel Protein kinase Cs (PKCs) in the pathophysiology of the aberrant megakaryocytopoiesis in PMF, concluding that the over-expression of PKCε detains a crucial relevance in the aberrant behavior of PMF megakaryocytes and its inhibition is capable to restore their normal differentiation and abrogate the anti-apoptotic signaling. Both results are discussed in the view of their therapeutic implications. In case PCM1/JAK2-related hematologic neoplasms, ERK-inhibitors rather than JAK-inhibitors (i.e. ruxolitinib) should be considered as a “tailored” drugs. In case of PMF, PKCε-inhibitors (i.e. εV1-2 peptide) configure as an appealing strategy to re-direct the megakaryocytic neoplastic clone.

Tear analysis and lens-tear interactions:part II. Ocular lipids-nature and fate of meibomian gland phospholipids

Purpose: Published data indicate that the polar lipid content of human meibomian gland secretions (MGS) could be anything between 0.5% and 13% of the total lipid. The tear film phospholipid composition has not been studied in great detail and it has been understood that the relative proportions of lipids in MGS would be maintained in the tear film. The purpose of this work was to determine the concentration of phospholipids in the human tear film. Methods: Liquid chromatography mass spectrometry (LCMS) and thin layer chromatography (TLC) were used to determine the concentration of phospholipid in the tear film. Additionally, an Amplex Red phosphatidylcholine-specific phospholipase C (PLC) assay kit was used for determination of the activity of PLC in the tear film. Results: Phospholipids were not detected in any of the tested human tear samples with the low limit of detection being 1.3 µg/mL for TLC and 4 µg/mL for liquid chromatography mass spectrometry. TLC indicated that diacylglycerol (DAG) may be present in the tear film. PLC was in the tear film with an activity determined at approximately 15 mU/mL, equivalent to the removal of head groups from phosphatidylcholine at a rate of approximately 15 µM/min. Conclusions: This work shows that phospholipid was not detected in any of the tested human tear samples (above the lower limits of detection as described) and suggests the presence of DAG in the tear film. DAG is known to be at low concentrations in MGS. These observations indicate that PLC may play a role in modulating the tear film phospholipid concentration.

The relationship between alexithymia and salivary cortisol levels in somatoform disorders

The purpose of this study was to investigate cortisol levels as a function of the hypothalamic-pituitary-adrenal axis (HPA) in relation to alexithymia in patients with somatoform disorders (SFD). Diurnal salivary cortisol was sampled in 32 patients with SFD who also underwent a psychiatric examination and filled in questionnaires (Toronto Alexithymia Scale, TAS scale; Screening for Somatoform Symptoms, SOMS scale; Hamilton Depression Scale, HAMD). The mean TAS total score in the sample was 55.69.6, 32% of patients being classified as alexithymic on the basis of their TAS scores. Depression scores were moderate (HAMD=13.2, Beck Depression Inventory, BDI=16.5). The patients' alexithymia scores (TAS scale Difficulty identifying feelings) correlated significantly positively with their somatization scale scores (Symptom Checklist-90 Revised, SCL-90-R); r=0.3438 (P0.05) and their scores on the Global Severity Index (GSI) on the SCL-90-R; r=0.781 (P0.01). Regression analysis was performed with cortisol variables as the dependent variables. Cortisol levels [measured by the area under the curve-ground (AUC-G), area under the curve-increase (AUC-I) and morning cortisol (MCS)] were best predicted in a multiple linear regression model by lower depressive scores (HAMD) and more psychopathological symptoms (SCL-90-R). No significant correlations were found between the patients' alexithymia scores (TAS) and cortisol levels. The healthy control group (n=25) demonstrated significantly higher cortisol levels than did the patients with SFD; in both tests P0.001 for AUC-G and AUC-I. However, the two groups did not differ in terms of their mean morning cortisol levels (P0.05). The results suggest that pre-existing hypocortisolism might possibly be associated with SFD.

Randomised masked clinical trial of the MGDRx eyebag for the treatment of meibomian gland dysfunction-related evaporative dry eye

Background/aims To investigate the efficacy and safety of the MGDRx EyeBag (The Eyebag Company, Halifax, UK) eyelid warming device. Methods Twenty-five patients with confirmed meibomian gland dysfunction (MGD)-related evaporative dry eye were enrolled into a randomised, single masked, contralateral clinical trial. Test eyes received a heated device; control eyes a non-heated device for 5 min twice a day for 2 weeks. Efficacy (ocular symptomology, noninvasive break-up time, lipid layer thickness, osmolarity, meibomian gland dropout and function) and safety (visual acuity, corneal topography, conjunctival hyperaemia and staining) measurements were taken at baseline and follow-up. Subsequent patient device usage and ocular comfort was ascertained at 6 months. Results Differences between test and control eyes at baseline were not statistically signi ficant for all measurements ( p>0.05). After 2 weeks, statistically significant improvements occurred in all efficacy measurements in test eyes ( p<0.05). Visual acuity and corneal topography were unaffected (p>0.05). All patients maintained higher ocular comfort after 6 months ( p<0.05), although the bene fit was greater in those who continued usage 1-8 times a month (p<0.001). Conclusions The MGDRx EyeBag is a safe and effective device for the treatment of MGD-related evaporative dry eye. Subjective benefit lasts at least 6 months, aided by occasional retreatment. Trial registration number NCT01870180.

Microwave decontamination of eyelid warming devices for the treatment of meibomian gland dysfunction

PURPOSE: The role of bacteria in meibomian gland dysfunction is unclear, yet contamination of compresses used as treatment may exacerbate this condition. This study therefore determined the effect of heating on bacteria on two forms of compress. METHODS: Cotton flannels and MGDRx EyeBags (eyebags) were inoculated by adding experimental inoculum (Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa; one species for each set of 3 eyebags and flannels). One of each were then randomised in to 3 groups: no heating (control); therapeutic (47.4±0.7°C); or sanitisation (68±1.1°C). After treatment, bacteria cell numbers were calculated. The experiment was repeated in triplicate. RESULTS: There was a statistically significant difference between each treatment with the eyebag for S. aureus (control=7.15±0.11logC/ml, therapeutic heating=5.24±0.59logC/ml, sanitisation heating=3.48±1.43logC/ml; P<0.001) and S. pyogenes (7.36±0.13, 5.73±0.26, 4.75±0.54; P<0.001). P. aeruginosa also showed a significant reduction (P<0.001) from control (6.39±0.34) to therapeutic (0.33±0.26) and sanitisation (0.33±0.21), but the latter were similar (P=1.000). For the flannels, there was significant difference between each treatment for S. aureus (6.89±0.46, 3.96±1.76, 0.42±0.90; P<0.001). For S. pyogenes, there was a significant reduction (P<0.001) from control (7.51±0.10) to therapeutic (5.91±0.62) and sanitisation (5.18±0.8), but the latter were similar (P=0.07). For P. aeruginosa, there was a significant difference (P<0.001) from control (7.15±0.36) to sanitisation (5.83±0.44); but not to therapeutic (6.84±0.31) temperatures (P=0.07). CONCLUSIONS: Therapeutic heating produces a significant reduction in bacteria on the eyebags, but only sanitisation heating appears effective for flannels. However, patients should be advised to heat the eyebag to sanitisation temperatures on initial use.

Isolation and characterization of the U1 and U2 snRNAs in the silk gland of the 5th instar larval stage silk moth Bombyx mori

In isolation and characterization studies, expression level U1 and U2 snRNA isoforms were obtained from the 5th instar larval stage silk gland (SG). The DNA content of the SG cells is approximately 200,000-fold higher compared to the usual (2N) somatic cells of B. mori due to endoreduplication. In this study, the existence of U1 and U2 snRNA isoforms in the SG of the organism is investigated. Bombyx mori U1 and U2-specific RT-PCR libraries from the silk gland were generated. Five U1 and eight U2 isoforms were isolated and characterized. Nucleotide differences, structural alterations, as well as protein and RNA interaction sites were analyzed in these variants. For the U1 snRNA variants, they were compared to the previously reported BmN isoforms. In all these U-snRNA variants, polymorphic sites do not predominate at the core of known functional sequences, which were interspecifically conserved. Variant sites and inter-species differences are located in moderately conserved regions. Free energy (ΔG) values for the entire U1 and U2 snRNA secondary structures and for the individual stem/loops domains of the isoforms were generated and compared to determine their structural stability. This will be the first time that U1 and U2 variants are shown specific for a development stage (larval) other than embryonic or adult. ^ Using phylogenetic analysis, evolutionary trees were generated for the U1 and U2 snRNAs using animal, plant, protista and fungal species. The resulting trees were boostrapped for robustness and rooted with the self-splicing RNA group II intron sequence from the cyanobacterium Calothrix. Using phylogenetic analyses, possible structural and functional evolutionary interdependence between the U1 and U2 snRNAs was investigated. ^

Thyroid hormone and gland status of Greenland sledge dogs (Canis lupus familiaris) exposed to contaminated blubber

As a model of high trophic level carnivores, sledge dogs were fed from 2 to 18 months of age with minke whale blubber containing organohalogen compounds (OHC) corresponding to 128 µg PCB/day. Controls were fed uncontaminated porcine fat. Thyroid hormone levels were assessed in 7 exposed and 7 control sister bitches (sampled at age 6-18 months) and 4 exposed and 4 control pups, fed the same diet as their mothers (sampled age 3-12 months). Lower free and total T3 and T4 were seen in exposed vs. control bitches beyond 10 months of age, and total T3 was lower through 3-12 months of age in exposed pups. A negative correlation with thyroid gland weight was significant for SumDDT, as was a positive association with total T3 for dieldrin. This study therefore supports observational data that OHCs may adversely affect thyroid functions, and it suggests that OHC exposure duration of 10 months or more may be required for current OHC contamination levels to result in detectable adverse effects on thyroid hormone dynamics.

Generation of an epigenetic signature by chronic hypoxia in prostate cells

Increasing levels of tissue hypoxia have been reported as a natural feature of the aging prostate gland and may be a risk factor for the development of prostate cancer. In this study, we have used PwR-1E benign prostate epithelial cells and an equivalently aged hypoxia-adapted PwR-1E sub-line to identify phenotypic and epigenetic consequences of chronic hypoxia in prostate cells. We have identified a significantly altered cellular phenotype in response to chronic hypoxia as characterized by increased receptor-mediated apoptotic resistance, the induction of cellular senescence, increased invasion and the increased secretion of IL-1 beta, IL6, IL8 and TNFalpha cytokines. In association with these phenotypic changes and the absence of HIF-1 alpha protein expression, we have demonstrated significant increases in global levels of DNA methylation and H3K9 histone acetylation in these cells, concomitant with the increased expression of DNA methyltransferase DMNT3b and gene-specific changes in DNA methylation at key imprinting loci. In conclusion, we have demonstrated a genome-wide adjustment of DNA methylation and histone acetylation under chronic hypoxic conditions in the prostate. These epigenetic signatures may represent an additional mechanism to promote and maintain a hypoxic-adapted cellular phenotype with a potential role in tumour development.

Transcriptional profiling of the oesophageal gland region of male worms of Schistosoma mansoni

The intestinal tract of schistosomes opens at the mouth and leads into the foregut or oesophageal region that is lined with syncytium continuous with the apical cytoplasm of the tegument. The oesophagus is surrounded by a specialised gland, the oesophageal gland. This gland releases materials into the lumen of the oesophagus and the region is thought to initiate the lysis of erythrocytes and neutralisation of immune effectors of the host. The oesophageal region is present in the early invasive schistosomulum, a stage potentially targetable by anti-schistosome vaccines. We used a 44k oligonucleotide microarray to identify highly up-regulated genes in microdissected frozen sections of the oesophageal gland of male worms of S. mansoni. We show that 122 genes were up-regulated 2-fold or higher in the oesophageal gland compared with a whole male worm tissue control. The enriched genes included several associated with lipid metabolism and transmembrane transport as well as some micro-exon genes. Since the oesophageal gland is important in the initiation of digestion and the fact that it develops early after invasion of the mammalian host, further study of selected highly up-regulated functionally important genes in this tissue may reveal new anti-schistosome intervention targets for schistosomiasis control.

The Calreticulin gene and myeloproliferative neoplasms

The Philadelphia negative myeloproliferative neoplasms include polycythaemia vera (PV), essential thrombocytopenia (ET) and primary myelofibrosis (PMF). Patients with these conditions were mainly thought to harbour JAK2V617F mutations or an Myeloproliferative leukaemia (MPL) substitution. In 2013, two revolutionary studies identified recurrent mutations in a gene that encodes the protein calreticulin (CALR). This mutation was detected in patients with PMF and ET with non-mutated JAK2 or MPL but was absent in patients with PV. The CALR gene encodes the calreticulin protein, which is a multifactorial protein, mainly located in the endoplasmic reticulum in chromosome 19 and regulates calcium homeostasis, chaperones and has also been implicated in multiple cellular processes including cell signalling, regulation of gene expression, cell adhesion, autoimmunity and apoptosis. Somatic 52 bp deletions and recurrent 52 bp insertion mutations in CALR were detected and all resulted in frameshift and clusters in exon 9 of the gene. This review will summarise the current knowledge on the CALR gene and mutation of the gene in pathological conditions and patient phenotypes.

MRI-based assessment of the pineal gland in a large population of children aged 0-5 years and comparison with pineoblastoma: part I, the solid gland.

INTRODUCTION: Differentiation between normal solid (non-cystic) pineal glands and pineal pathologies on brain MRI is difficult. The aim of this study was to assess the size of the solid pineal gland in children (0-5 years) and compare the findings with published pineoblastoma cases. METHODS: We retrospectively analyzed the size (width, height, planimetric area) of solid pineal glands in 184 non-retinoblastoma patients (73 female, 111 male) aged 0-5 years on MRI. The effect of age and gender on gland size was evaluated. Linear regression analysis was performed to analyze the relation between size and age. Ninety-nine percent prediction intervals around the mean were added to construct a normal size range per age, with the upper bound of the predictive interval as the parameter of interest as a cutoff for normalcy. RESULTS: There was no significant interaction of gender and age for all the three pineal gland parameters (width, height, and area). Linear regression analysis gave 99 % upper prediction bounds of 7.9, 4.8, and 25.4 mm(2), respectively, for width, height, and area. The slopes (size increase per month) of each parameter were 0.046, 0.023, and 0.202, respectively. Ninety-three percent (95 % CI 66-100 %) of asymptomatic solid pineoblastomas were larger in size than the 99 % upper bound. CONCLUSION: This study establishes norms for solid pineal gland size in non-retinoblastoma children aged 0-5 years. Knowledge of the size of the normal pineal gland is helpful for detection of pineal gland abnormalities, particularly pineoblastoma.