Successful Multidisciplinary Management of Aortic Valve Repair in Severe Hemophilia B with Extended Half-Life Recombinant Factor IX Concentrate.

Successful management of surgery in severe coagulation disorders depends on adequate replacement of the deficient factors from intervention until wound healing. Extended half-life (EHL) recombinant factor IX (rFIX) has been increasingly used in hemophilia B (HB) patients. Monitoring of blood levels of EHL rFIX allows to obtain pharmacokinetic (PK) parameters in order to optimize and personalize therapeutic scheme. We describe a case of a young male with severe HB who successfully underwent aortic valve repair. This is the first reported open-heart surgery in a patient with severe HB using EHL rFIX. The success was based on accurate PK evaluation and on meticulous preoperative planning and close cooperation among surgeons, hemophilia specialists, and laboratory team despite the long distance between hemophilia center and surgical clinic.

Long term changes in health complaints after removal of amalgam restorations.

OBJECTIVE: Concerns over adverse effects of mercury released from dental amalgam sometimes lead patients to request removal of their amalgam restorations. Several studies report improvement of subjective health after removal of amalgam restorations, but the mechanisms are unclear. The aim of this paper is to present data on long term changes in intensity of health complaints after amalgam removal in a group of patients with health complaints self-attributed to dental amalgam. Data from the five years follow-up in a clinical trial are presented and related to potential determinants of change. MATERIALS AND METHODS: Patients previously referred to a specialty unit for health complaints attributed to amalgam restorations were included in the study. The 20 participants who were allocated to the treatment group had all amalgam restorations removed and replaced with other dental restorative materials. Intensity of health complaints was calculated from questionnaire data and personality variables were measured by MMPI-2. RESULTS: At the follow-up five years after the amalgam removal was completed, intensity of general health complaints was significantly reduced (p=.001), but the symptom load was still high. The reduction was significantly correlated with concentration of mercury in urine at pre-treatment. There were no significant correlations with personality variables. CONCLUSIONS: Removal of amalgam restorations was followed by a long term reduction of general health complaints, which was associated with mercury concentration in urine before amalgam removal. Additional studies are needed to confirm the potential mechanisms for the observed reduction.

Somatic, hematologic phenotype, long-term outcome, and effect of hematopoietic stem cell transplantation. An analysis of 97 Fanconi anemia patients from the Italian national database on behalf of the Marrow Failure Study Group of the AIEOP (Italian Association of Pediatric Hematology-Oncology).

We analyzed 97 Fanconi anemia patients from a clinic/biological database for genotype, somatic, and hematologic phenotype, adverse hematological events, solid tumors, and treatment. Seventy-two patients belonged to complementation group A. Eighty percent of patients presented with mild/moderate somatic phenotype and most with cytopenia. No correlation was seen between somatic/hematologic phenotype and number of missense mutations of FANCA alleles. Over follow-up, 33% of patients improved or maintained mild/moderate cytopenia or normal blood count, whereas remaining worsened cytopenia. Eleven patients developed a hematological adverse event (MDS, AML, pathological cytogenetics) and three developed solid tumors. 10 years cumulative risk of death of the whole cohort was 25.6% with median follow-up 5.8 years. In patients eligible to hematopoietic stem cell transplantation because of moderate cytopenia, mortality was significantly higher in subjects transplanted from matched unrelated donor over nontransplanted subjects, whereas there was no significant difference between matched sibling donor transplants and nontransplanted patients. In patients eligible to transplant because of severe cytopenia and clonal disease, mortality risk was not significantly different in transplanted from matched unrelated versus matched sibling donor versus nontransplanted subjects. The decision to transplant should rely on various elements including, type of donor, HLA matching, patient comorbidities, impairment, and clonal evolution of hematopoiesis. Am. J. Hematol. 91:666-671, 2016. © 2016 Wiley Periodicals, Inc.

Mycophenolate mofetil and Sirolimus as second or further line treatment in children with chronic refractory Primitive or Secondary Autoimmune Cytopenias: a single centre experience.

The management of refractory autoimmune cytopenias in childhood is challenging due to the lack of established evidence on escalating treatments. The long-term efficacy of immunosuppressive drugs was evaluated in children with refractory autoimmune cytopenias referred to the Haematology Unit of the Gaslini Children's Hospital between 2001 and 2014. Patients were grouped into three categories: autoimmune lymphoproliferative syndrome (ALPS), ALPS-related syndrome (at least one absolute/primary additional criterion for ALPS) and primary autoimmune cytopenia (PAC, cytopenia with no other immunological symptoms/signs). Fifty-eight children (aged 1-16 years) entered the study: 12 were categorized with ALPS, 24 were ALPS-related and 22 had PAC. Five didn't receive treatment. Fifty-three were initially treated with steroids/intravenous immunoglobulin. Fourteen responded, whereas 39 did not. Of these 39 patients, 34 (87%) received mycophenolate mofetil (MMF) as second/further-line treatment and 22 (65%) responded. Within these 34 subjects, ALPS patients responded better (11/11, 100%) than the two other groups pooled together (11/23, 48%; P = 0·002). Sirolimus was given as second/further-line treatment to 16 children, and 12 (75%) responded, including 8 who previously failed MMF therapy. Median follow-up was 3·46 years. MMF and Sirolimus were well-tolerated and enabled partial/complete and sustained remission in most children. These drugs may be successfully and safely used in children with refractory autoimmune cytopenias with or without ALPS/ALPS-related disorders and may represent a valid second/further line option.

Diagnosis and management of acquired aplastic anemia in childhood. Guidelines from the Marrow Failure Study Group of the Pediatric Haemato-Oncology Italian Association (AIEOP).

Acquired aplastic anemia (AA) is a rare heterogeneous disease characterized by pancytopenia and hypoplastic bone marrow. The incidence is 2-3/million inhabitants/year, in Europe, but higher in East Asia. Survival in severe aplastic anemia (SAA) has markedly improved in the past 2 decades because of advances in hematopoietic stem cell transplantation, immunosuppressive and biologic drugs, and supportive care. In SAA hematopoietic stem cell transplant (HSCT) from a matched sibling donor (MSD) is the treatment of choice. If a MSD is not available, the options include immunosuppressive therapy (IST) or unrelated donor HSCT. The objective of this guideline is to provide healthcare professionals with clear guidance on the diagnosis and management of pediatric patients with AA. A preliminary, evidence-based document issued by a group of pediatric hematologists was discussed, modified and approved during a series of "Consensus Conferences" according to procedures previously validated by the AIEOP Board. The guidelines highlight the importance of referring pediatric patients with AA to pediatric centers with long experience in diagnosis, differential diagnosis, management, supportive care and follow-up of AA.

Allogeneic hematopoietic stem cell transplantation in Fanconi anemia: the European Group for Blood and Marrow Transplantation experience.

Although allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative treatment for patients with Fanconi anemia (FA), published series mostly refer to single-center experience with limited numbers of patients. We analyzed results in 795 patients with FA who underwent first HSCT between May 1972 and January 2010. With a 6-year median follow-up, overall survival was 49% at 20 years (95% confidence interval, 38-65 years). Better outcome was observed for patients transplanted before the age of 10 years, before clonal evolution (ie, myelodysplastic syndrome or acute myeloid leukemia), from a matched family donor, after a conditioning regimen without irradiation, the latter including fludarabine. Chronic graft-versus-host disease and secondary malignancy were deleterious when considered as time-dependent covariates. Age more than 10 years at time of HSCT, clonal evolution as an indication for transplantation, peripheral blood as source of stem cells, and chronic graft-versus-host disease were found to be independently associated with the risk for secondary malignancy. Changes in transplant protocols have significantly improved the outcome of patients with FA, who should be transplanted at a young age, with bone marrow as the source of stem cells.

Impaired immune response to Candida albicans in cells from Fanconi anemia patients.

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure and cancer predisposition. Several studies show alterations of the immunological status of FA patients including defects in peripheral blood lymphocyte subsets, serum immunoglobulin levels, and inflammatory cytokines. However scanty information is available on the response of FA cells to specific infectious antigens. In this work we examined the response of FA cells to different immunological stimuli and found a defective response of IL-1ß, TNF-α and IL-17 to Candida albicans stimulation thus pointing to a potentially impaired response to fungal infections of FA patients.

Clinical aspects of Fanconi anemia individuals with the same mutation of FANCF identified by next generation sequencing.

BACKGROUND: Fanconi anemia (FA) is a rare genetic disease characterized by congenital malformations, aplastic anemia and increased risk of developing malignancies. FA is genetically heterogeneous as it is caused by at least 17 different genes. Among these, FANCA, FANCC, and FANCG account for approximately 85% of the patients whereas the remaining genes are mutated in only a small percentage of cases. For this reason, the molecular diagnostic process is complex and not always extended to all the FA genes, preventing the characterization of individuals belonging to rare groups. METHODS: The FA genes were analyzed using a next generation sequencing approach in two unrelated families. RESULTS: The analysis identified the same, c.484_485del, homozygous mutation of FANCF in both families. A careful examination of three electively aborted fetuses in one family and one affected girl in the other indicated an association of the FANCF loss-of-function mutation with a severe phenotype characterized by multiple malformations. CONCLUSION: The systematic use of next generation sequencing will allow the recognition of individuals from rare complementation groups, a better definition of their clinical phenotypes, and consequently, an appropriate genetic counseling.

p38 MAPK inhibition suppresses the TLR-hypersensitive phenotype in FANCC- and FANCA-deficient mononuclear phagocytes.

Fanconi anemia, complementation group C (FANCC)-deficient hematopoietic stem and progenitor cells are hypersensitive to a variety of inhibitory cytokines, one of which, TNFα, can induce BM failure and clonal evolution in Fancc-deficient mice. FANCC-deficient macrophages are also hypersensitive to TLR activation and produce TNFα in an unrestrained fashion. Reasoning that suppression of inhibitory cytokine production might enhance hematopoiesis, we screened small molecules using TLR agonist-stimulated FANCC- and Fanconi anemia, complementation group A (FANCA)-deficient macrophages containing an NF-κB/AP-1-responsive reporter gene (SEAP). Of the 75 small molecules screened, the p38 MAPK inhibitor BIRB 796 and dasatinib potently suppressed TLR8-dependent expression of the reporter gene. Fanconi anemia (FA) macrophages were hypersensitive to the TLR7/8 activator R848, overproducing SEAP and TNFα in response to all doses of the agonist. Low doses (50nM) of both agents inhibited p38 MAPK-dependent activation of MAPKAPK2 (MK2) and suppressed MK2-dependent TNFα production without substantially influencing TNFα gene transcription. Overproduction of TNFα by primary FA cells was likewise suppressed by these agents and involved inhibition of MK2 activation. Because MK2 is also known to influence production and/or sensitivity to 2 other suppressive factors (MIP-1α and IFNγ) to which FA hematopoietic progenitor cells are uniquely vulnerable, targeting of p38 MAPK in FA hematopoietic cells is a rational objective for preclinical evaluation.

Molecular analysis of Fanconi anemia: the experience of the Bone Marrow Failure Study Group of the Italian Association of Pediatric Onco-Hematology.

Fanconi anemia is an inherited disease characterized by congenital malformations, pancytopenia, cancer predisposition, and sensitivity to cross-linking agents. The molecular diagnosis of Fanconi anemia is relatively complex for several aspects including genetic heterogeneity with mutations in at least 16 different genes. In this paper, we report the mutations identified in 100 unrelated probands enrolled into the National Network of the Italian Association of Pediatric Hematoly and Oncology. In approximately half of these cases, mutational screening was carried out after retroviral complementation analyses or protein analysis. In the other half, the analysis was performed on the most frequently mutated genes or using a next generation sequencing approach. We identified 108 distinct variants of the FANCA, FANCG, FANCC, FANCD2, and FANCB genes in 85, 9, 3, 2, and 1 families, respectively. Despite the relatively high number of private mutations, 45 of which are novel Fanconi anemia alleles, 26% of the FANCA alleles are due to 5 distinct mutations. Most of the mutations are large genomic deletions and nonsense or frameshift mutations, although we identified a series of missense mutations, whose pathogenetic role was not always certain. The molecular diagnosis of Fanconi anemia is still a tiered procedure that requires identifying candidate genes to avoid useless sequencing. Introduction of next generation sequencing strategies will greatly improve the diagnostic process, allowing a rapid analysis of all the genes.

Sirolimus as maintenance treatment in an infant with life-threatening multiresistant pure red cell anemia/autoimmune hemolytic anemia.

A 9-month-old boy with life-threatening multiresistant pure red cell anemia/autoimmune hemolytic anemia within the frame of a possible, undiagnosed immune-mediated disease was initially treated with prednisone. Further-line therapies of the following 7 relapses included immunoglobulins, rituximab, cyclophosphamide, and alentuzumab followed by other maintenance treatments as cyclosporine, methotrexate, and mycophenolate. After all the administered therapies failed, the patient was successfully treated by splenectomy followed by fludarabine and then sirolimus as maintenance treatment. Relapses might have been caused by the lack of a complete debulking of triggering cells and/or ineffective maintenance therapy. Splenectomy and sirolimus may have played a complementary role in the management of both situations.

Prolongated Activated Partial Thromboplastin Time (aPTT) in Pediatric Patients before Surgery-Crying Wolf: Lupus (Anticoagulant) Does Not Always Threaten Children.

A prolonged preoperatory aPTT in children is often the cause of a delay of scheduled surgeries and the repetition of multiple blood tests, with the consequent wasting of resources and significant discomfort for children and parents. The aim of this review is to analyze the situations in which an isolated prolongation of aPTT is found during preoperative evaluation in children, especially when it is due to the presence of antiphospholipid antibodies, providing the readers with the keys to interpret this situation and the possibility to correctly evaluate the hemorrhagic risk of a patient.

Immunological profile of Fanconi anemia: a multicentric retrospective analysis of 61 patients.

In this study, the immunological status of 61 patients with Fanconi anemia (FA) with advanced marrow failure before hematopoietic stem cell transplantation was analyzed by assessing the phenotype of peripheral blood lymphocytes, serum immunoglobulin (Ig) levels, and inflammatory cytokines. In patients with FA, total absolute lymphocytes (P < 0.0001), B cells (P < 0.0001), and NK cells (P = 0.003) were reduced when compared with normal controls. T cells (CD3), that is, cytotoxic T cells, naïve T cells, and regulatory T cells, showed a relative increase when compared with controls. Serum levels of IgG (P < 0.0001) and IgM (P = 0.004) were significantly lower, whereas IgA level was higher (P < 0.0001) than in normal controls. TGF-ß (P = 0.007) and interleukin (IL)-6 (P = 0.0007) levels were increased in the serum of patients when compared with controls, whereas sCD40L level decreases (P < 0.0001). No differences were noted in the serum levels of IL-1ß, IL-2, IL-4, IL-10, IL-13, IL-17, and IL-23 between FA subjects and controls. This comprehensive immunological study shows that patients with FA with advanced marrow failure have an altered immune status. This is in accordance with some characteristics of FA such as the proinflammatory and proapoptotic status. In addition, B lymphocyte failure may make tight and early immunological monitoring advisable.

Characterization of health complaints before and after removal of amalgam fillings--3-year follow-up.

OBJECTIVE: Some patients attribute health complaints to amalgam fillings and report improvement of health after replacement of amalgam fillings. The aim of the present study was to characterize the changes of different health complaints after replacement of amalgam fillings and compare with an external reference group from the general population. MATERIALS AND METHODS: The study group included 20 patients with health complaints attributed to amalgam fillings who were participants in the treatment group of a clinical trial at the Norwegian Dental Biomaterials Adverse Reaction Unit. The patients were asked to indicate the intensity of local and general health complaints on numeric rating scales (0-10) before removal of amalgam fillings and at follow-up 3 years after removal. Data from the patient group were compared with data from an external reference group (n = 441). RESULTS: Before treatment the mean intensity of complaints were on a higher level in the treatment group compared to the reference group. The most frequently reported complaints in the treatment group were gastrointestinal symptoms, fatigue, pain from muscles and joints, symptoms from ear/nose/throat and difficulty concentrating. From pre-treatment examination to the 3-year follow-up 20 of 23 health complaints decreased, being statistically significant for taste disturbances, pain from muscles and joints, gastrointestinal complaints, complaints from ear/nose/throat and fatigue. CONCLUSIONS: The inter-individual variation of intensities of health complaints was considerable and the reduction of health complaints varied for the different complaints. Several factors may be of importance for the observed reduction of complaint intensity.

High frequency of ribosomal protein gene deletions in Italian Diamond-Blackfan anemia patients detected by multiplex ligation-dependent probe amplification assay.

Diamond-Blackfan anemia is an autosomal dominant disease due to mutations in nine ribosomal protein encoding genes. Because most mutations are loss of function and detected by direct sequencing of coding exons, we reasoned that part of the approximately 50% mutation negative patients may have carried a copy number variant of ribosomal protein genes. As a proof of concept, we designed a multiplex ligation-dependent probe amplification assay targeted to screen the six genes that are most frequently mutated in Diamond-Blackfan anemia patients: RPS17, RPS19, RPS26, RPL5, RPL11, and RPL35A. Using this assay we showed that deletions represent approximately 20% of all mutations. The combination of sequencing and multiplex ligation-dependent probe amplification analysis of these six genes allows the genetic characterization of approximately 65% of patients, showing that Diamond-Blackfan anemia is indisputably a ribosomopathy.

Detection and quantification of monomers in unstimulated whole saliva after treatment with resin-based composite fillings in vivo.

Resin-based dental restorative materials contain allergenic methacrylate monomers, which may be released into saliva after restorative treatment. Monomers from resin-based composite materials have been demonstrated in saliva in vitro; however, studies analyzing saliva after restorative therapy are scarce. The aim of this study was to quantify methacrylate monomers in saliva after treatment with a resin-based composite filling material. Saliva was collected from 10 patients at four start points--before treatment, and 10 min, 24 h, and 7 d after treatment--and analysed by combined chromatography/mass spectrometry. The monomers bisphenol-A diglycidyl methacrylate (Bis-GMA), 2-hydroxyethyl methacrylate (HEMA), and urethane dimethacrylate (UDMA) were detected and quantified in the samples collected shortly (10 min) after treatment. The amounts detected ranged from 0.028 to 9.65 µg ml(-1) for Bis-GMA, from 0.015 to 0.19 µg ml(-1) for HEMA, and from 0.004 to 1.2 µg ml(-1) for UDMA. Triethyleneglycol dimethacrylate (TEGDMA) was detected in four of the samples. Ethoxylated bisphenol-A dimethacrylate (Bis-EMA) was not detected. Monomers were not detected in saliva samples collected before treatment, or 24 h or 7 d after treatment, with the exception of one sample, 24 h after treatment, in which HEMA was detected. In conclusion, monomers from the investigated resin-based composite and adhesive system were present in saliva shortly after treatment. One week after treatment, no monomers could be detected in patients' saliva samples.

TLR8-dependent TNF-(alpha) overexpression in Fanconi anemia group C cells.

Tumor necrosis factor alpha (TNF-alpha) production is abnormally high in Fanconi anemia (FA) cells and contributes to the hematopoietic defects seen in FA complementation group C-deficient (Fancc(-/-)) mice. Applying gene expression microarray and proteomic methods to studies on FANCC-deficient cells we found that genes encoding proteins directly involved in ubiquitinylation are overrepresented in the signature of FA bone marrow cells and that ubiquitinylation profiles of FA-C and complemented cells were substantially different. Finding that Toll-like receptor 8 (TLR8) was one of the proteins ubiquitinylated only in mutant cells, we confirmed that TLR8 (or a TLR8-associated protein) is ubiquitinylated in mutant FA-C cells and that TNF-alpha production in mutant cells depended upon TLR8 and the canonical downstream signaling intermediates interleukin 1 receptor-associated kinase (IRAK) and IkappaB kinase-alpha/beta. FANCC-deficient THP-1 cells and macrophages from Fancc(-/-) mice overexpressed TNF-alpha in response to TLR8 agonists but not other TLR agonists. Ectopically expressed FANCC point mutants were capable of fully complementing the mitomycin-C hypersensitivity phenotype of FA-C cells but did not suppress TNF-alpha overproduction. In conclusion, FANCC suppresses TNF-alpha production in mononuclear phagocytes by suppressing TLR8 activity and this particular function of FANCC is independent of its function in protecting the genome from cross-linking agents.

Bivalirudin anticoagulation to overcome heparin resistance in a neonate with cerebral sinovenus thrombosis.

: Anticoagulation in a neonate is a challenge and the availability of anticoagulant options is extremely limited. Here we describe the use of a direct thrombin inhibitor, bivalirudin, in a full-term neonate with symptomatic cerebral sinovenous thrombosis complicated by bilateral thalamic hemorrhagic stroke and intraventricular hemorrhage, who could not be effectively treated with sodium heparin due to heparin resistance (HR) and showed thrombosis regression after start of bivalirudin treatment, without worsening of the hemorrhage. While the use of bivalirudin in neonates has been previously described, the indication of cerebral sinovenous thrombosis and the setting of HR are unique.

Rituximab-based immunosuppression for autoimmune haemolytic anaemia in infants.

We report a case series of four infants with severe autoimmune haemolytic anaemia (AIHA) who responded to treatment with rituximab and cyclosporine after having failed first line therapy with high-dose steroid (prednisolone 4-8 mg/kg/d). Rituximab was started at 11-90 d from onset due to continued haemolysis; three infants also received cyclosporine A. Three of four infants reached complete response, defined as normal haemoglobin, reticulocytes and negative indices of haemolysis, at 7-21 months from diagnosis. In long-term follow-up two infants remained disease-free with normal immunology, one had undefined immunodeficiency and one had autoimmune lymphoproliferative syndrome.