Sonia Cannella

Novel Munc13-4 mutations in children and young adult patients with haemophagocytic lymphohistiocytosis.

Familial haemophagocytic lymphohistiocytosis (FHL) is a genetically heterogeneous disorder characterised by constitutive defects in cellular cytotoxicity resulting in fever, hepatosplenomegaly and cytopenia, and the outcome is fatal unless treated by chemoimmunotherapy followed by haematopoietic stem-cell transplantation. Since 1999, mutations in the perforin gene giving rise to this disease have been identified; however, these account only for 40% of cases. Lack of a genetic marker hampers the diagnosis, suitability for transplantation, selection of familial donors, identification of carriers, genetic counselling and prenatal diagnosis. Mutations in the Munc13–4 gene have recently been described in patients with FHL. We sequenced the Munc13–4 gene in all patients with haemophagocytic lymphohistiocytosis not due to PRF1 mutations. In 15 of the 30 families studied, 12 novel and 4 known Munc13–4 mutations were found, spread throughout the gene. Among novel mutations, 2650C→T introduced a stop codon; 441del A, 532del C, 3082del C and 3226ins G caused a frameshift, and seven were mis sense mutations. Median age of diagnosis was 4 months, but six patients developed the disease after 5 years of age and one as a young adult of 18 years. Involvement of central nervous system was present in 9 of 15 patients, activity of natural killer cells was markedly reduced or absent in 13 of 13 tested patients. Chemo-immunotherapy was effective in all patients. Munc13–4 mutations were found in 15 of 30 patients with FHL without PRF1 mutations. Because these patients may develop the disease during adolescence or even later, haematologists should include FHL2 and FHL3 in the differential diagnosis of young adults with fever, cytopenia, splenomegaly and hypercytokinaemia.

Germline mutations of the perforin gene are a frequent occurrence in childhood anaplastic large cell lymphoma

Monoallelic and biallelic mutations of the PRF1 gene have been reported in some cases of childhood lymphoma. Anaplastic large cell lymphoma (ALCL) accounts for 10% to 15% of all childhood lymphomas. To assess the possible role of PRF1 mutations in ALCL, the authors screened a series of patients collected by the Associazione Italiana di Ematologia Oncologia Pediatrica (AIEOP).

Mutations affecting mRNA splicing are the most common molecular defect in patients with familial hemophagocytic lymphohistiocytosis type 3

This report shows that UNC13D mutations leading to splicing errors are frequent in hemophagocytic lymphohisti-ocytosis type 3. Their detection and identification may be instrumental in the diagnosis of this disease. Mutations of UNC13D have been described in patients affected by familial hemophagocytic lymphohistiocytosis (FHL3). The Munc13-4 protein contributes to the priming of the secretory granules. Mutation in this gene results in defective cellular cytotoxicity and the familial hemophagocytic lymphohistiocytosis clinical picture. Among reported mutations, few are predicted to impair splicing. Yet, functional impact of these mutations has not been addressed. We identified 18 out of 31 familial hemophagocytic lymphohistiocytosis families showing at least one mutation responsible for splicing error. We identified some known and three novel splicing mutations: one falls at the acceptor site of exon 11 and 2 are deep intronic mutations in IVS1 and in IVS30. We demonstrated that these deep intronic mutations affect regulatory sequences causing aberrant splicing. We report that UNC13D mutations leading to splicing errors represent the majority of mutations observed in familial hemophagocytic lymphohistiocytosis. This finding has implications for designing strategies for analysis of the families with suspected familial hemophagocytic lymphohistiocytosis.

Altered mRNA expression of PAX5 is a common event in acute lymphoblastic leukaemia

transformation. Regarding late haematological toxicity, one of our eight patients developed bone marrow hypoplasia with pancytopenia 7 months after completion of 4 cycles of FCR, which was complicated by pneumonia. This was successfully treated with i.v. antibiotics, neutrophil counts recovered to normal with temporary G-CSF administration and the patient is now transfusionand growth factor-independent in ongoing CR (haemoglobin 110 g/l, white cell count 3Æ3 · 10/l, neutrophil count 1Æ73 · 10/l, platelet count 68 · 10/l now 10 months post-completion of four cycles of FCR). Delayed haematological toxicity associated with fludarabinebased chemotherapy is well-reported from chronic lymphocytic leukaemia trials (Tam et al, 2008). We have also observed delayed haematological recovery unrelated to persistent disease in 43% of patients with indolent lymphoproliferative diseases treated with fludarabine combinations (Gill et al, 2009). Also, the combination of fludarabine with alkylators results in a modest risk of development of myelodysplasia that may be higher than with fludarabine alone (Tam et al, 2006). Therefore, patients require careful follow-up after completion of therapy and prompt investigation and treatment with appropriate antimicrobials for febrile episodes. Thorough monitoring provided, in our experience this potential complication is manageable in the majority of cases and should not preclude patients with MZL being considered for treatment with one of the most effective therapies for their disease.

Vocal cord palsy after vincristine treatment in a child and the inefficacy of glutamic acid in the prevention of relapse: a case report

IntroductionVincristine is an antineoplastic drug with a well known efficacy for the treatment of acute lymphoblastic leukemia and many solid tumors. No more than 20 pediatric patients with vincristine-induced vocal cord palsy have been reported, and to the best of our knowledge this is the first case where glutamic acid was administered with the aim of preventing a relapse of laryngeal dysfunction.Case presentationThe larynx paralysis presented with hoarseness and stridor in a Caucasian 18-month-old girl and spontaneously resolved in about a month. In order to administer a subsequent full dose of vincristine, our patient received oral glutamic acid whose efficacy against vincristine neurological side effects has been previously reported.ConclusionsSince in our patient the amino acid proved to be ineffective in the prevention of laryngeal paralysis relapse, we suggest that a dose reduction of vincristine should be preferred by oncologists as an initial approach after a case of drug-induced vocal cord palsy.

The spectrum of non-classical Diamond-Blackfan anemia: a case of late beginning transfusion dependency associated to a new RPL5 mutation

Diamond Blackfan anemia typically presents in infants and is often associated with many kinds of malformations. Severity of anemia often needs transfusional support in the first months of life. We describe here a patient with Diamond Blackfan anemia related to a RPL5 mutation. The patient had no physical abnormalities and experienced a very late onset of transfusion dependency.

TRANSPLANTATION OF UNMANIPULATED ALLOGENEIC PBSC : PRELIMINARY REPORT ON 24 PATIENTS

To explore the feasibility and potential advantages of PBSC in allogeneic transplantation, we grafted 24 patients (age 16-57, median 37) with different hematologic diseases (ALL = 10, AML = 5, MM = 4, NHL = 2, CML = 1, MDS = 1, AA = 1), 23 HLA-identical to their siblings and 1 partially matched. Cells were collected from donors by apheresis after G-CSF 10 to 16 mg/kg/day for 4 to 5 days, and stored at 4 degrees C until infusion. The patients were conditioned with chemotherapy regimens including busulfan and cyclophosphamide in the majority of cases and received GVHD prophylaxis with CSA-MTX in all but two. The graft consisted of PBSC alone, with a median of 143.5 (range 18.1-358.9) x 10(4)/kg CFU-GM, 9.0 (range 3.3-18.0) x 10(6)/kg CD34+ cells and 2.8 (range 1.2 to 8.6) x 10(8)/kg CD3+ and cells. An ANC >0.0.5 x 10(9)/L was recovered on (median) day 13 (range 11-17), and a platelet count >50 x 10(9)/L on (median) day 13 (range 12-55) post graft. There was no correlation between CD34+ cells or CFU-GM number in the inoculum and time to hematologic reconstitution. Acute GVHD (grade II-IV) occurred in 10 out of 22 (45%), chronic GVHD in 10 out of 18 evaluable (55%) patients. We found no relationship between occurrence of acute or chronic GVHD and number of CD3+ cells in the graft. Four patients relapsed and 7 died after transplantation. Fifteen patients are currently alive and disease-free 67 to 710 (median 286) days from the graft. Allogeneic transplantation with unmanipulated PBSC ensures a fast and stable engraftment. Acute GVHD incidence and severity seems comparable to that of bone marrow transplantation, but there may be an increase in chronic GVHD, mainly of the extensive form.

Upregulation of miR-29a and genomic DNA hypermethylation in normal karyotype AML showing DNMT3A mutation

A hydrocarbons have attracted concern as a kind of environmental pollutants along with possible health threats. Comamonas testosteroni strain P19 was originally isolated as a biphenyl degrader and capable of degrading several aromatic compounds as a sole source of carbon and energy. Here, we describe the metabolic insight by genetic and physiological analysis indicating how aromatic compounds are metabolized in C. testosteroni strain P19. The strain has a circular chromosome of 5,633,218 bp with a G+C content of 67.65% and one plasmid. The annotation identified gene clusters encoding essential proteins for metabolizing several aromatic hydrocarbons including anthranilate, benzoate, biphenyl, m-hydroxybenzoate, p-hydroxybenzoate, p-methoxybenzoate, phenol, phthalate, protocatechuate, terephthalate etc. Random plasposon mutagenesis confirmed that the predicted gene clusters were essential in the metabolic pathways of the compounds. In addition, we identified novel metabolite on degradation of ferulate which had very similar structure to vanillate. Based on structural assignment, the unknown product was revealed as a dimer of vanillate that was a novel intermediate in ferulate degradation pathway. Subsequently, genomic approach suggested the metabolic network of aromatic compounds in C. testosteroni strain P19.S enterica serovar typhimurium (Salmonella typhimurium) is the most common causative agent of human gastroenteritis after consumption of contaminated seafood. The use of lytic bacteriophages against this pathogen can be a new and promising approach for the prevention of food-contamination and food-borne infection. This study investigated the potential application of the bacteriophage SE-5 during depuration to reduce S. typhimurium in cockles (Cerastoderma edule) at different multiplicity of infection (MOI). Cockles were infected with 106 colony-forming units (CFU)/mL of S. typhimurium in the seawater and each infected group was treated with four different MOI values: 100, 10, 1 and 0.1. Infected cockles were depurated in non-recirculating seawater at 16°C for 12 hour. After S. typhimurium accumulation at 16° C, the initial mean values of bacteria in cockles were 6.20 log CFU/g. Depuration with phages at MOI 0.1 was the best condition to inactivate S. typhimurium in cockles, the concentration was reduced by 1.7 log CFU/g after 2 hours of depuration. Reduction for the other MOI values (MOI=1; MOI=10; MOI=100) was 1.13, 1.21 and 2.10 log CFU/g after 6 hours, 12 hours and 12 hours of treatment respectively. To our knowledge, this is the first report of a depuration trial using bacteriophage in the cockle treatment process. The data of this study indicate clearly that the application of the bacteriophages could reduce significantly the population of S. typhimurium in infected cockles. Moreover, cockles could be maintained alive during the depuration process. Therefore, the application of bacteriophage was effectively proven to be useful for shellfish depuration.S members of the genus Lavandula (Lavenders) are economically important crop plants that produce and store large quantities of essential oils (EO; a mixture of primarily monoterpenes) in glandular trichomes, or oil glands. Because glandular trichomes are specialized for EO production, they strongly express the related structural and regulatory genes. We have generated substantial genomic resources to probe essential oil metabolism in lavenders, and have cloned several key EO biosynthetic genes including lavandulyl diphosphate synthase, caryophyllene, borneol dehydrogenase, cineole synthase, and beta-phellandrene synthase from these plants. We have also developed a set of EST-SSR markers capable of delineating a number of lavender species effectively. In this presentation the author will present a summary of his recent findings.This novel, albeit abstract representation of alphaIntroduction The superfamily of 2OG-dependent dioxygenases, by definition, comprises members from all major taxa, and participates in neutralizing the effects of several forms of abiotic stress, viz., pesticides, hypoxia, and osmotic. The oxidative decarboxylation of 2-oxoglutarate to succinate is coupled with a concomitant substrate hydroxylation and, is in most cases, followed by an additional specialized conversion.W optics has historically been mainly used for imaging applications, light has now become a tool of manipulation and direct interaction with biological samples. It is well known that when light impinges on an object, it exerts a small pressure on it known as optical force. An immediate application of optical forces is found in optical tweezers, which in biology are usually employed as a non-invasive technique to immobilize cellular organisms for imaging purposes. These techniques, including more pioneering ideas such as, e.g., cell optical sorting and in-vivo manipulation, require a precise understanding on how the optical pressure affects biological organisms. This calls for the development of precise numerical methods, whose development will be crucial for applications of the aforementioned techniques. In this respect, ab-initio techniques represent a very important tool that can provide quantitative answers to the problem. By employing a series of massively parallel FDTD simulations, we study how optical forces act on biological matter. As a representative case study, we here consider deformed Red Blood Cells (RBC) illuminated by a monochromatic plane wave. We consider typical deformations arising due to the presence of a disease such as malaria. Realistic parameters for the geometry and the refractive index are then taken from published experiments. In our theoretical campaign, we study the dependence of the optical force on the particle shape and the incident wavelength. We show that optical forces change appreciably with the deformation, with amplitude variation in the pN range for RBCs affected by disease.Background: Epidermal Growth Factor receptor (EGFR) family is a group of four highly conserved trans-membrane tyrosine kinase receptors (EGFR, ErbB2/HER2, ErbB3/HER3 and ErbB4/HER4) involved in fate decision of many pivotal biological processes. Several mutations on EGFR have been associated to number of human cancers making it one of the most sought after target for cancer therapeutics. However despite its therapeutic importance, its full length structural behavior is still illusive. Therefore to utilize and exploit this magic drug target, it is imperative to decipher its structure.Most immunosuppressive agents were initially developed as antibiotics produced by the genus Streptomyces . This investigation was devoted to explore the bioactive metabolite of the Streptomyces variabilis ASU319 extract and testing the purified active compound of this extract as an immunosuppressive agent in rats blood. Elucidation of the chemical structure and optimization of the active compound were studied as well. Antimicrobial activity was conducted using agar-well diffusion and disc diffusion assays. The antimicrobial metabolite was extracted from the fermentation broth by ethyl acetate and purified by TLC and silica gel column chromatography. The pure active compound was then subjected to spectroscopic analyses: 1 H NMR, Elemental analysis, IR and Mass spectra. The active antimicrobial compound was tested as an immunosuppressive agent by injection in the rat blood and the complete blood count (CBC) was determined. The crude extracts of the selected active antagonistic five isolates were tested to prevent the inflammation and proliferation of lymphocytes of the rats blood. The active antimicrobial compound of Streptomyces ASU319 was purified and proven as an immunosuppressive agent. The tested compound decreased each of the neutrophils, lymphocytes and monocytes than the positive control. The compound was of molecular weight 458 g/mol and had given the proposed chemical formula C 24 H 46 O 8 . The most potent bacterial isolate was identified by 16SrRNA sequencing as Streptomyces variabilis ASU319 with accession number [GenBank: KC145278]. These results revealed that Streptomyces variabilis ASU319 is potential microbial for production of active antimicrobial compound that has the ability to decrease the proliferation of the lymphocytes cells in the blood and may be a good immunosuppressive agent.New fluorescent N-(2 and 3-methoxyphenyl) thieno [3, 2-b] pyridin-7-amines were synthesized by C-N coupling of 7-bromothieno [3, 2-b] pyridine with 2 or 3 methoxy anilines and gave very low growth inhibitory (GI50) values when studied in human tumor cell lines but also in non-tumor cells. So, pursuing a future clinical administration of these compounds, they were encapsulated in nanoliposomes due to the toxicity presented in vitro in non-tumor cells. The compounds are reasonably fluorescent in solvents of different polarity exhibiting fluorescence quantum yields between 10% and 60%. Nanoliposomes are technological developments for the encapsulation and delivery of bioactive agents. Because of their biocompatibility and biodegradability along with their size, nanoliposomes have potential applications in a vast range of fields including nanotherapy. Nanoliposomes are able to enhance the performance of bioactive agents by improving their bioavailability in vitro and in vivo stability as well as preventing their unwanted interactions with other molecules. These compounds have been successfully encapsulated in different nanoliposome formulations as revealed by fluorescence emission and fluorescence anisotropy measurements. These results are important for future drug delivery developments using these compounds as antitumor agents.H infections are present worldwide and microorganisms such as viruses, bacteria, fungi and parasites can be responsible for such infections. Fungal infections exhibit low occurrence; however, because they take longer to detect and treat, high rates of morbidity and mortality are known. Thus, fungal infections namely those associated to Aspergillus niger represent actual challenges especially for elderly, neonates or those having an underlying disease or under immunosuppressive drug treatments. Microbial metabolomics has been breaking new ground as a very useful tool in several areas including those related to microbial diagnosis. Microorganisms produce several volatile metabolites, the whole of which can be used as unique chemical fingerprints of each species and possibly of strain. This richness of information holds the promise for diagnosing infections. This research study aims to in-depth study the A. niger exometabolome in order to establish metabolites pattern that can be further exploited for fungal diagnosis. A methodology based on advanced multidimensional gas chromatography (HSSPME/GC×GC-ToFMS) tandem with multivariate analysis was developed. Different growth conditions were assayed. A. niger exometabolome revealed around 500 metabolites distributed over several chemical families being the major ones alcohols, aldehydes, esters, hydrocarbons, ketones and terpenoids. A subset of 44 metabolites defined as the A. niger metabolomic biomarkers pattern was selected and successfully used to distinguish this species from others previously chosen revealing its useful potential.T cell phone technology is an integral part of everyday life and, it is used to chat including voice and picture and in addition has many other applications. Male reproductive system is highly compartmentalized and sensitive biological system that requires the integration of intrinsic and extrinsic factors to normal function, hence we studied effects of GMS (Towers and Mobile radiation) on reproductive hormones (FSH, AMH, LH, Testosterone) and interference of other factors such as smoking, distant and hours near towers, mobile using period, and tumors related with. Our study was applied on 144 samples and was compared with 50 controls. The result showed significant increase in LH & FSH levels while significant decrease in Testosterone & AMH hormone, also there was a significant increase in hormone levels of smoking and nonsmoking persons and distend from tower and a large percentage of tumor disease.M pulegium and Eucalyptus camaldulensis are important plant species with applications in flavouring processed foods. Their essential oils were tested for antifungal activity to explore biocontrol ways vs. fungal rot of apples in storage. Extracts analyses by gas chromatography-mass spectrometry revealed M. pulegium dominated by pulegone and E. camaldulensis, by 1,8 cineole and α pinene. Oils antifungal activity was studied vs. Alternaria alternata and Penicillium expansum. Oil of M. pulegium is thrice most active than E. camaldulensis. Chemical components show synergism. Inhibition suggests food-preservation application. Classification algorithms are proposed based on information entropy and its production. Oils components are classified by numbers of C=C bonds, O atoms and cycles. Classification algorithms are based on information entropy. When applying procedures to moderate-size sets, excessive results appear compatible with data and suffer combinatorial explosion; however, after equipartition conjecture one obtains selection criterion resulting from classification between hierarchical trees. Information entropy permits classifying oils components and agrees with principal component analyses.H metals are common environmental pollutants to the aquatic organisms. Several aquatic species have been used as biomarkers and bio-monitoring subjects for heavy metal pollution. Behavioral changes are sensitive markers of toxicity. The effects of Cd and Mn on the survival, locomotion, attachment and feeding behaviors of the gastropod snail Biomphalaria alexandrina were determined. The 96-h LC50 for Cd and Mn were 0.38 and 156.57 mg/l, respectively. Snails were exposed to sub-lethal concentrations of each metal plus a control for chronic exposure period (16-20 days) and the survival curves were determined. Locomotion and feeding behaviors of snails exposed to Cd and Mn at acute (96 h exposure) and chronic exposure (24 days exposure) intervals were recorded. The survival curves showed that long term exposure of snails to ascending concentrations of Cd and Mn caused a gradual decrease in the survival rate of B. alexandrina in a dose-dependent manner. Compared to control, a significant decrease was recorded in the feeding and locomotion behaviors of exposed snails. The tendency to feed in B. alexandrina was significantly decreased by acute exposure to Cd and completely blocked by Mn exposure. The feeding rate was 4.8±0.68 bites/min. in Cd-exposed snails compared to 16.3±1.7 bites/minute in control. A significant decrease was recorded in the locomotion behavior of exposed snail groups compared to control. The path length of traces was 3351.2±95.3 mm in control compared to 295.3±50 and 1610.5±58.9 mm, respectively for Mn and Cd exposed snails. Thus, sub-lethal metal concentrations affected B. alexandrina behaviors, potentially impacting the snail’s activity and tendency to feed. The present study also demonstrated B. alexandrina as a sensitive bio-indicator and can be used as a model organism to assess heavy metals risk factors for severe toxicity in freshwater ecosystems.1 (ACC) deaminase promotes plant growth by sequestering and cleaving the ethylene precursor ACC to α-ketobutyrate and ammonium. Many plant growth promoting rhizobacteria producing 1-aminocyclopropane-1-carboxylate (ACC) deaminase as a source of nitrogen has an eminent role in plant nutrition. The present work deals with comparative analysis of ACCD producing plant growth-promoting rhizobacteria (PGPR) are Azospirillum lipoferum, Phyllobacterium brassicacearum, Pseudomonas fluorescens, Francisella tularensis subsp. holarctica OSU18 and Bacillus cereus. The sequence and phylogenetic analysis of ACCD producing PGPR species represents the common conserved domain belonging to the tryptophan synthase beta subunit-like PLP-dependent enzymes super family and closely related to each other. The predicted homology models of ACCD of PGPR have similar protein structure with similar folds often share similar function. This analysis represents the evolutionary conservation and same biochemical function of ACCD producing plant growth-promoting rhizobacteria. This analysis is very helpful to understand the biological function of PGPR species.Today, cell phone technology is an integral part of everyday life and, its use to chat including voice and picture in addition to many other applications. Male reproductive system is highly compartmentalized and sensitive biological system that requires the integration of intrinsic and extrinsic factors to normal function, hence we studied effects of GMS (Towers and Mobil radiation)on reproductive hormones (FSH,AMH,LH, Testosterone) and interference of other factors such as smoking ,distant and hours near towers, Mobil using period, and tumors related with .Our study applied on (144) sample compared with (50) control, the result showed significant increase in LH&FSH levels while significant decrease in Testosterone & AMH hormone, also significant increase inhormone levels smoking and non-smoking persons and distend from tower.S steroid hormones regulate multiple female reproductive functions. Cyclooxygenase-2 (COX-2) is an inflammationassociated enzyme to regulate prostaglandin production. Previous studies demonstrated that COX-2-deficient mice failed to ovulate suggesting a vital role of COX-2 in ovulation. Sex steroid hormones were reported to modulate COX-2 expression: Dihydrotestosterone (DHT) was able to inhibit interlukin-1β-induced COX-2 expression in vascular smooth muscle cells while estradiol (E2) was able to promote COX-2 expression in the rat oviduct. In the ovary, the significance and the involving mechanisms of androgens and estrogens in COX-2 regulation remain mostly unclear. The aim of this study was to clarify whether and how sex steroid hormones affect COX-2 expression in rat ovarian granulosa cells. Previous studies suggested that PKC could be activated by FSH or LH in follicular granulosa cells leading to inflammatory-like consequences. Thus, a PKC activator PDD (phorbol-12, 13-didecanoate) was used in this study. It was noted that DHT appeared to attenuate PDDinduced COX-2 protein, mRNA expression and promoter activity; However, E2 was able to enhance PDD induced COX-2 protein, mRNA expression and promoter activity. In addition, the PDD-mediated PGE2 production was also impacted by DHT and E2. The PDD-mediated COX-2 expression was inhibited by parthenolide (NF-κB inhibitor) but was enhanced by SP600125 (JNK inhibitor) or wortmannin (PI3K inhibitor). Thus, DHT and E2 may affect PKC-mediated inflammation in ovarian granulosa cells by acting through these signaling players.D to the natural complementary properties in DNA sequences based on double helix pair exploring longer DNA pieces and its reverse fragment in complementary symmetry with a distance play an important role in modern genetic analysis. In this talk, the substring complementary string matching technique is used to detect and analyze the structure of DNA motif. We focused on the relationship between fragment and its reverse fragment in complementary symmetry and the possible structure of palindromic sequences, hairpin prediction. Statistical measurements are used to process some selected human genome data and all possible corresponding fragments are identified. We convert the identified data into visualized figures to illustrate the visible analysis results of quantities of complex DNA sequences as extracted information.