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 disorder characterized by pancytopenia and hypoplastic bone marrow. The incidence is 2-3 per million population per year in the Western world, but 3 times higher in East Asia. Survival in severe aplastic anemia (SAA) has improved significantly due to advances in hematopoietic stem cell transplantation (HSCT), immunosuppressive therapy, biologic agents, and supportive care. In SAA, HSCT from a matched sibling donor (MSD) is the first-line treatment. If a MSD is not available, options include immunosuppressive therapy (IST), matched unrelated donor, or haploidentical HSCT. The purpose of this guideline is to provide health care professionals with clear guidance on the diagnosis and management of pediatric patients with AA. A preliminary evidence-based document prepared by a group of pediatric hematologists of the Bone Marrow Failure Study Group of the Italian Association of Pediatric Hemato-Oncology (AIEOP) was discussed, modified and approved during a series of consensus conferences that started online during COVID 19 and continued in the following years, according to procedures previously validated by the AIEOP Board of Directors.

Disruption of Toxoplasma gondii-Induced Host Cell DNA Replication Is Dependent on Contact Inhibition and Host Cell Type.

The protozoan Toxoplasma gondii is a highly successful obligate intracellular parasite that, upon invasion of its host cell, releases an array of host-modulating protein effectors to counter host defenses and further its own replication and dissemination. Early studies investigating the impact of T. gondii infection on host cell function revealed that this parasite can force normally quiescent cells to activate their cell cycle program. Prior reports by two independent groups identified the dense granule protein effector HCE1/TEEGR as being solely responsible for driving host cell transcriptional changes through its direct interaction with the cyclin E regulatory complex DP1 and associated transcription factors. Our group independently identified HCE1/TEEGR through the presence of distinct repeated regions found in a number of host nuclear targeted parasite effectors and verified its central role in initiating host cell cycle changes. Additionally, we report here the time-resolved kinetics of host cell cycle transition in response to HCE1/TEEGR, using the fluorescence ubiquitination cell cycle indicator reporter line (FUCCI), and reveal the existence of a block in S-phase progression and host DNA synthesis in several cell lines commonly used in the study of T. gondii. Importantly, we have observed that this S-phase block is not due to additional dense granule effectors but rather is dependent on the host cell line background and contact inhibition status of the host monolayer in vitro. This work highlights intriguing differences in the host response to reprogramming by the parasite and raises interesting questions regarding how parasite effectors differentially manipulate the host cell depending on the in vitro or in vivo context. IMPORTANCE Toxoplasma gondii chronically infects approximately one-third of the global population and can produce severe pathology in immunologically immature or compromised individuals. During infection, this parasite releases numerous host-targeted effector proteins that can dramatically alter the expression of a variety of host genes. A better understanding of parasite effectors and their host targets has the potential to not only provide ways to control infection but also inform us about our own basic biology. One host pathway that has been known to be altered by T. gondii infection is the cell cycle, and prior reports have identified a parasite effector, known as HCE1/TEEGR, as being responsible. In this report, we further our understanding of the kinetics of cell cycle transition induced by this effector and show that the capacity of HCE1/TEEGR to induce host cell DNA synthesis is dependent on both the cell type and the status of contact inhibition.

Characterization of children with FLT3-ITD acute myeloid leukemia: a report from the AIEOP AML-2002 study group.

Recurrent molecular markers have been routinely used in acute myeloid leukemia (AML) for risk assessment at diagnosis, whereas their post-induction monitoring still represents a debated issue. We evaluated the prognostic value and biological impact of minimal residual disease (MRD) and of the allelic ratio (AR) of FLT3-internal-tandem duplication (ITD) in childhood AML. We retrospectively screened 494 children with de novo AML for FLT3-ITD mutation, identifying 54 harboring the mutation; 51% of them presented high ITD-AR at diagnosis and had worse event-free survival (EFS, 19.2 versus 63.5% for low ITD-AR, <0.05). Forty-one percent of children with high levels of MRD after the 1st induction course, measured by a patient-specific real-time-PCR, had worse EFS (22.2 versus 59.4% in low-MRD patients, P<0.05). Next, we correlated these parameters with gene expression, showing that patients with high ITD-AR or persistent MRD had characteristic expression profiles with deregulated genes involved in methylation and acetylation. Moreover, patients with high CyclinA1 expression presented an unfavorable EFS (20.3 versus 51.2% in low CyclinA1 group, P<0.01). Our results suggest that ITD-AR levels and molecular MRD should be considered in planning clinical management of FLT3-ITD patients. Different transcriptional activation of epigenetic and oncogenic profiles may explain variability in outcome among these patients, for whom novel therapeutic approaches are desirable.

Can the presence of cat/dog at home be considered the only criterion of exposure to cat/dog allergens? A likely underestimated bias in clinical practice and in large epidemiological studies.

An important aspect of allergic sensitization to furry animals is the association of dog and cat exposure in early childhood with the incidence of respective allergies later in life. This topic is very controversial, because some authors have found a "facilitating" effect, while others have noticed a "protective" or even no significant effect in individuals living in urban areas. It is likely that some biases could be responsible of these contradictory findings. Cat/dog ownership or their presence in indoor environments are considered usually the main criteria to assess the exposure to these pets in studies' questionnaires. Even in clinical practice "are there animals at home?" is the common query usually done when collecting anamnestic data. In our opinion, these commonly used questions should not be considered the main index of exposure to pet allergens, because they can lead to erroneous interpretation of the clinical significance of positive skin prick tests for pet allergens as well as of the real risk of exposure to allergens of dog/cat in epidemiological studies. Consequently, we suggest a new, more realistic, classification of modalities of exposure to pet allergens in "real life" based on five possible conditions.

The isochromosome i(7)(q10) carrying c.258+2t>c mutation of the SBDS gene does not promote development of myeloid malignancies in patients with Shwachman syndrome.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder, characterized by exocrine pancreatic insufficiency, skeletal abnormalities and bone marrow (BM) dysfunction with an increased risk to develop myelodysplastic syndrome and/or acute myeloid leukaemia (MDS/AML). SDS is caused, in nearly 90% of cases, by two common mutations (that is, c.183_184TA>CT and c.258+2T>C) in exon 2 of the SBDS gene, localized on chromosome 7. Clonal chromosome anomalies are often found in the BM of SDS patients; the most frequent is an isochromosome for long arms of chromosome 7, i(7)(q10). We studied eight patients with SDS carrying the i(7)(q10) who were compound heterozygotes for SBDS mutations. By assessing the parental origin of the i(7)(q10) using microsatellite analysis, we inferred from the results which mutation was present in double dose in the isochromosome. We demonstrate that in all cases the i(7)(q10) carries a double dose of the c.258+2T>C, and we suggest that, as the c.258+2T>C mutation still allows the production of some amount of normal protein, this may contribute to the low incidence of MDS/AML in this subset of SDS patients.

The polymorphisms -318C>T in the promoter and 49A>G in exon 1 of CTLA4 and the risk of aplastic anemia in a Caucasian population.

Aplastic anemia (AA) is a rare disease with a major autoimmune pathogenetic component. CTLA4 is a T-lymphocyte surface molecule involved in the maintenance of immune tolerance. Some polymorphisms associated with a reduced expression of CTLA4, and thus presumably with increased tendency to autoimmunity, have been associated with various autoimmune diseases. In this study, we evaluated the distribution of the low expression polymorphisms -318C > T and 49A > G of CTLA4 in a population of 67 patients with acquired AA and in 100 normal controls. There was no difference in the distribution of the tested polymorphism between patients and controls and, within the patient group, between those who responded to immunosuppression vs those who did not respond. This study indicates that the polymorphisms -318C > T and 49A > G of CTLA4 do not affect the risk of developing AA and do not influence the response to immunosuppression.

Relationship between Bet v 1 and Bet v 2 specific IgE and food allergy in children with grass pollen respiratory allergy.

Grass allergy is the most common pollinosis in Northern Italy. Some patients with grass allergy show polysensitization against other pollens and plant-derived foods. In these patients oral allergic syndrome (OAS) is frequently associated. To evaluate the correlation between food allergy or food sensitization and specific IgE against panallergens such as Bet v 1 and Bet v 2, we studied 56 children (mean age: 8 years 5 months) suffering from respiratory allergy due to grass pollens were enrolled. Specific IgE against the most important food, inhalant allergen and Bet v 1, Bet v 2 were performed by ImmunoCAP technology (UniCAP 1000, Pharmacia Diagnostics, Uppsala, Sweden). We found 14 children (25%) sensitized to Bet v 1 and 13 (23%) to Bet v 2; in 24 cases (42.3%) a sensitization to at least one of the 2 panallergens was observed. Five of the 14 cases (36%) sensitized to Bet v 1 showed food allergy and 8 (57%) food sensitization; 6 (46%) of the 13 children sensitized to Bet v 2 showed food allergy and 7 (54%) food sensitization; only one case of Bet v 1 specific IgE without food allergy or sensitization was seen. Sixteen subjects (29%) showed food allergy (group A); 20 children (35.5%) multiple sensitizations to inhalant and at least one plant-derived food (group B); 20 subjects (35.5%) only inhalant allergens (group C). Sensitization to Bet v 1 (P<0.03) and Bet v 2 (P<0.009) is from a statistical point of view significantly higher in groups A and B than in group C. In the 16 patients with food allergy hazelnut was the major triggering food (50%), followed by peanut (38%), kiwi (31%), apple and walnut (19%). Specific IgE for Bet v 1 is more associated with nuts and legumes, while Bet v 2 is more related to fresh fruit and vegetables. In conclusion grass pollinosis is frequently associated with polysensitization to other pollen and food allergens. Bet v 1 and Bet v 2 specific IgE are significantly higher in these patients than in patients with grass monosensitization, and this sensitization may be considered a possible risk factors to evolve later into food allergy. Among the offending foods, legumes and the nut group are mostly related to Bet v 1, while vegetables and fresh fruits to Bet v 2.

Clinical effectiveness of a silk fabric in the treatment of atopic dermatitis.

BACKGROUND: In children with atopic dermatitis (AD), eczema is easily aggravated by contact with irritant factors (e.g. aggressive detergents, synthetic and woollen clothes, climatic factors). OBJECTIVES: To evaluate the effectiveness of a special silk fabric (MICROAIR DermaSilk) in the treatment of young children affected by AD with acute lesions at the time of examination. METHODS: Forty-six children (mean age 2 years) affected by AD in an acute phase were recruited: 31 received special silk clothes (group A) which they were instructed to wear for a week; the other 15 served as a control group (group B) and wore cotton clothing. Topical moisturizing creams or emulsions were the only topical treatment prescribed in both groups. The overall severity of the disease was evaluated using the SCORAD index. In addition, the local score of an area covered by the silk clothes was compared with the local score of an uncovered area in the same child. All patients were evaluated at baseline and 7 days after the initial examination. RESULTS: At the end of the study a significant decrease in AD severity was observed in the children of group A (mean SCORAD decrease from 43 to 30; P = 0.003). At the same time, the improvement in the mean local score of the covered area (from 32 to 18.6; P = 0.001) was significantly greater than that of the uncovered area (from 31 to 26; P = 0.112). CONCLUSIONS: The use of special silk clothes may be useful in the management of AD in children.

A comparison of different allergometric tests, skin prick test, Pharmacia UniCAP and ADVIA Centaur, for diagnosis of allergic diseases in children.

BACKGROUND: The diagnosis of allergic disease is performed by skin prick tests (SPT) or through the demonstration of specific IgE in a blood sample via an in vitro test. The measurement of IgE concentration against allergens provides critical information in clinical allergy. Standardized and reproducible methods contribute to the quality of diagnosis and treatment of allergic disease. METHODS: In this study we evaluated the performance of a new specific IgE method, developed by ALK-Abellò for Bayer Diagnostics to run on their ADVIA Centaur immunoassay system. One hundred and fifty-one children with allergic diseases (both food and inhalant allergies) were tested for specific IgE (sIgE) via SPT and in vitro tests (UniCAP system, Pharmacia, and ADVIA Centaur immunoassay system, Bayer Diagnostics) and the test results were correlated with the clinical data. RESULTS: Statistical analysis revealed no significant difference between the two in vitro tests compared with clinical history. The sensitivities and specificities are similar, but the UniCAP system method has higher sensitivity. In the children with cow's milk allergy, the UniCAP system has sensitivity of 91% and specificity of 70%; the ADVIA Centaur immunoassay has sensitivity of 82% and specificity of 74%. In hen's egg allergy, UniCAP system has 94% sensitivity and 64% specificity, and the ADVIA Centaur system has 88% sensitivity and 52% specificity. In inhalant allergies, the two methods show statistically similar performances for both grass pollen allergies (UniCAP sensitivity 100%, specificity 73%; ADVIA Centaur sensitivity 95%, specificity 79%) and in the dust mites allergies (UniCAP sensitivity 91%, specificity 62%; ADVIA Centaur sensitivity 86%, specificity 64%). In cat allergies, the systems showed equivalent results (UniCAP sensitivity 100%, specificity 71%; ADVIA Centaur sensitivity 100%, specificity 70%). Using the UniCAP system, the geometric mean of sIgE values in children with clinical allergy is significantly higher than in sensitized ones. The ADVIA Centaur system shows a similar trend with the exclusion of cow's milk and Dermatophagoides farinae allergens. With this last method the mean value of sIgE is higher in sensitized than in symptomatic children. CONCLUSION: The new ADVIA Centaur method compares favorably with the results obtained on the UniCAP system. If other studies continue to confirm this data, then the advantages are numerous: the use of only a small quantity of serum (25 micro l per allergen), rapid turnaround time, minimal hands-on time, and no interference from IgG.

Extracellular pH changes and accompanying cation shifts during ouabain-induced spreading depression.

Interstitial ionic shifts that accompany ouabain-induced spreading depression (SD) were studied in rat hippocampal and cortical slices in the presence and absence of extracellular Ca(2+). A double-barreled ion-selective microelectrode specific for H(+), K(+), Na(+), or Ca(2+) was placed in the CA1 stratum radiatum or midcortical layer. Superfusion of 100 microM ouabain caused a rapid, negative, interstitial voltage shift (2-10 mV) after 3-5 min. The negativity was accompanied by a rapid alkaline transient followed by prolonged acidosis. In media containing 3 mM Ca(2+), the alkalosis induced by ouabain averaged 0.07 +/- 0.01 unit pH. In media with no added Ca(2+) and 2 mM EGTA, the alkaline shift was not significantly different (0.09 +/- 0.02 unit pH). The alkaline transient was unaffected by inhibiting Na(+)-H(+) exchange with ethylisopropylamiloride (EIPA) or by blocking endoplasmic reticulum Ca(2+) uptake with thapsigargin or cyclopiazonic acid. Alkaline transients were also observed in Ca(2+)-free media when SD was induced by microinjecting high K(+). The late acidification accompanying ouabain-induced SD was significantly reduced in Ca(2+)-free media and in solutions containing EIPA. The ouabain-induced SD was associated with a rapid but relatively modest increase in [K(+)](o). In the presence of 3 mM external Ca(2+), the mean peak elevation of [K(+)](o) was 12 +/- 0.62 mM. In Ca(2+)-free media, the elevation of [K(+)](o) had a more gradual onset and reached a significantly larger peak value, which averaged 22 +/- 1.1 mM. The decrease in [Na(+)](o) that accompanied ouabain-induced SD was somewhat greater. The [Na(+)](o) decreased by averages of 40 +/- 7 and 33 +/- 3 mM in Ca(2+) and Ca(2+)-free media, respectively. In media containing 1.2 mM Ca(2+), ouabain-induced SD was associated with a substantial decrease in [Ca(2+)](o) that averaged 0.73 +/- 0. 07 mM. These data demonstrate that in comparison with conventional SD, ouabain-induced SD exhibits ion shifts that are qualitatively similar but quantitatively diminished. The presence of external Ca(2+) can modulate the phenomenon but is irrelevant to the generation of the SD and its accompanying alkaline pH transient. Significance of these results is discussed in reference to the propagation of SD and the generation of interstitial pH changes.

Role of glycemia in acute spinal cord injury. Data from a rat experimental model and clinical experience.

While experimental and clinical evidence indicates that in brain injury blood glucose increases with injury severity and hyperglycemia worsens neurological outcome, the role of blood glucose in secondary mechanisms of neuronal damage after acute spinal cord injury has not yet been investigated. Data from spinal cord ischemia models suggests a deleterious effect of hyperglycemia, likely due to enhanced lactic acidosis, which is primarily dependent on the amount of glucose available to be metabolized. The purpose of this study is to summarize preliminary experimental and clinical observations on the role of blood glucose in acute spinal cord injury. Between 1995 and 1996 we used the New York University (NYU) rat spinal cord injury model to test the following hypotheses: 1) Blood glucose levels increase with injury severity. 2) Fasting protects from hyperglycemia and prevents secondary damage to the spinal cord. 3) Postinjury-induced hyperglycemia (dextrose 5% 2 gm/Kg) enhances spinal lesion volume. From a clinical perspective, we reviewed blood glucose records of 47 patients admitted to the Department of Neurosrgery in Verona, between 1991 and 1995, within 24 hours of acute spinal cord injury in order to determine: a) the incidence of hyperglycemia (> 140 mg/dl); b) the correlation between blood glucose and injury severity; and c) the role of methylprednisolone in affecting blood glucose. Results indicate that in a graded spinal cord injury model: 1) Early after injury, more severe contusions support significantly higher blood glucose levels. 2) Fasting overnight does not directly affect spinal cord lesion volume but influences blood gases, and we observed that a slightly systemic acidosis plays a minor neuroprotective role. Fasting also ensures more consistent normoglycemic baseline blood glucose values. 3) Postinjury-induced moderate hyperglycemia (160-190 mg/dl) does not significantly affect spinal cord injury. In the clinical study, we observed that during the first 24 hours after spinal cord injury: a) Glycemia ranges between 90 and 243 mg/dl (mean value 143 mg/dl), and close to 50% of the patients present blood glucose values higher than normal. b) Methylprednisolone administration is not associated to significantly higher blood glucose levels. c) There is a trend for larger glucose rises with more severe injury.

Xenotransplantation of transgenic oligodendrocyte-lineage cells into spinal cord-injured adult rats.

Spinal cord trauma is associated not only with loss of nerve cells and fibers but also with damage to oligodendrocytes and demyelination. In order to assess the potential of transplanted oligodendrocyte-lineage cells to repair the demyelination that follows spinal cord injury, we have used donor glia derived from a transgenic mouse line containing the LacZ transgene under control of the myelin basic protein promoter. Glia derived from fetal or neonatal transgenic mice were injected into the spinal cords of immunosuppressed adult rats at the site of an experimental traumatic lesion 1-16 days after injury. Cells expressing LacZ were identified 15-18 days later in cryosections rostral and caudal to the transplant site, most conspicuously within white matter defects. Some of these cells within the dorsal columns gave rise to approximately 30- to 60-microns processes, consistent with myelin segments, which are oriented parallel to the fiber tract. Glial transplantation may thus be a feasible means of replacing damaged host oligodendrocytes with donor oligodendrocyte-lineage cells capable of reforming myelin and potentially restoring functional lost as a result of demyelination associated with spinal cord injury.

Familial erythrophagocytic lymphohistiocytosis: adverse prognostic significance of delayed diagnosis.

Familial erythrophagocytic lymphohistiocytosis (FEL) is a rare disorder of the monocyte-macrophage system, for which an autosomal recessive mode of inheritance has been postulated. It is characterized by a dismal prognosis and is peculiar of early infancy. Three new cases of infants affected by FEL are reported. All three patients were diagnosed about three months after the onset of symptoms, and all three died shortly after diagnosis. The need for early diagnosis and prompt, intensive cytotoxic chemotherapy is emphasized.

Myoelectric evoked potentials versus locomotor recovery in chronic spinal cord injured rats.

The purpose of this study was to determine the utility of descending evoked potentials in evaluating functional recovery in rats after spinal cord contusion injury. Rats received thoracic contusions at T9 using a controlled-displacement impactor. They were evaluated for 5 weeks postinjury using auditory startle responses (ASR) while alert, or by cerebellar motor evoked potentials (CMEP) while anesthetized. ASR and CMEP were recorded electromyographically from forelimb and hindlimb muscles. Open field locomotor performance was also assessed and recovered to almost normal levels by 3 weeks postinjury. Histologic analysis of the injury site indicated that the contusions destroyed approximately 70% of the cross-sectional area of the cord. Although the remaining 30% was sufficient to preserve nearly normal locomotor behavior, ASR and CMEP amplitudes in hindlimb flexors and extensors were reduced by 90% or more after injury and showed virtually no recovery. Significant ASR and CMEP responses were present in the cutaneous trunk muscles of the lower torso after injury. These muscles are innervated via peripheral nerves originating at cord levels above the injury. Multi-wave field potentials normally recorded from the dorsal cord surface in response to cerebellar stimulation were absent in injured rats, suggesting minimal if any activation of segmental neurons via the pathways normally mediating CMEP. The tracts mediating ASR and CMEP thus appear to be highly sensitive to mild spinal cord trauma but are evidently not essential for support or walking.

Multidisciplinary treatment of primary orbital rhabdomyosarcoma. A single-institution experience.

Orbital rhabdomyosarcoma accounts for one-fourth of the primary tumors in the head and neck region. Modern treatment modalities have led to a 2-year survival rate of about 90% in these patients. However, new therapeutic trials are designed to reduce complications and salvage more than 90% of orbital cases. Between 1979 and 1990, 12 children affected by primary orbital rhabdomyosarcoma have been diagnosed and treated at the University of Naples. Ten of them have been uniformly treated by biopsy, followed by immediate radiation and combined chemotherapy. All 12 patients are alive and free of detectable disease, from a minimum of 7 months to a maximum of 123 months after diagnosis. In all children, ocular structures have been spared and the complications observed until now have been few. The above results suggest that the association of immediate radiation therapy and chemotherapy might represent an optimal tool for treatment of orbital rhabdomyosarcoma.

Non-Hodgkin's lymphoma in childhood presenting as thyroid enlargement.

The authors report a case of a 10-year-old girl with early involvement of the thyroid gland by non-Hodgkin's lymphoma, an uncommon site of presentation of childhood lymphomas. In pediatrics, thyroid enlargement is more often caused by lymphocytic thyroiditis. The good response to therapy, in spite of the advanced stage of the disease, is noted.