Anna Bossi

Restoration of CFTR function in patients with cystic fibrosis carrying the F508del-CFTR mutation

Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in CftrF508del homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the F508del-CFTR mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from F508del homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both in vivo, in mice, and in vitro, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 F508del-CFTR homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells in vivo, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of TNF/TNF-alpha (tumor necrosis factor) and CXCL8 (chemokine [C-X-C motif] ligand 8) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the F508del-CFTR mutation.

The European Consensus on grading of bone marrow fibrosis allows a better prognostication of patients with primary myelofibrosis

We investigated the relationship between the International Prognostic Scoring System of the International Working Group for Myelofibrosis Research and Treatment and the European Consensus on grading of bone marrow fibrosis (MF) in patients with primary myelofibrosis. We compared them in 196 consecutive primary myelofibrosis patients (median follow-up 45.7 months; range 7.4–159). International Prognostic Scoring System classified 42 cases as low risk, 73 as intermediate risk-1, 69 as intermediate risk-2, and 12 as high risk; European Consensus on grading of bone marrow fibrosis classified 83 cases as MF-0, 58 as MF-1, 41 as MF-2, and 14 as MF-3. By the time of the analysis, 30 patients (15.3%) had died. Overall median survival was 3.8 years (95% confidence interval: 3.3–4.3). Multivariate analysis confirmed that both scoring systems independently predicted survival, with hazard ratios similar to those provided by univariate analysis (respectively, 2.40 (95% confidence interval: 1.47–3.91) and 2.58 (95% confidence interval: 1.72–3.89) but the likelihood ratio increased from 19.6 of the International Prognostic Scoring System or 29.0 of the European Consensus on grading of bone MF to 42.3 when both measures were considered together. Analysis of the overall survival curves documented that patients classified as having the most favourable rate with both prognostic scores (ie low risk and MF-0) survive longer than those with only one favourable score (ie low risk but MF >0 or MF-0, but International Prognostic Scoring System >low risk). In contrast, those patients classified as having the most unfavourable rate for both scores (high risk and MF-3) have a shorter survival than those with only one unfavourable score (ie high risk but MF<3 or MF-3, but International Prognostic Scoring System <high risk). In conclusion, our analysis suggests that better prognostication can be achieved in primary myelofibrosis patients when both systems are used together.

Fetal growth velocity: kinetic, clinical, and biological aspects.

With the aim of determining fetal growth kinetics, prenatal data were analysed which had been longitudinally collected in the framework of a perinatal growth survey. The sample comprised 238 singleton normal pregnancies, selected in Genoa and Turin (between 1987 and 1990), and repeatedly assessed by ultrasound scans (five to nine per pregnancy). Five morphometric traits were considered: BPD (biparietal diameter), OFD (occipitofrontal diameter), HC (head circumference), FDL (femur diaphysis length) and AC (abdomen circumference). Growth rate seemed to increase in the early part of the second trimester, and decrease subsequently: velocity peaks were steeper and earlier for head diameters and circumference (about 18 weeks) than for femur length (20 weeks) and abdomen circumference (22 weeks). Velocity standards were traced using a longitudinal two-stage linear model: this ensures unbiased description of the shape of the growth curve, even when growth kinetics are asynchronous, and efficient estimation of the outer centiles--the most useful for diagnostic purposes.

Prothrombotic Gene Mutations in Patients with Sudden Sensorineural Hearing Loss and Cardiovascular Thrombotic Disease

Objectives: Impaired cochlear perfusion seems to be an important event in sudden sensorineural hearing loss. Prothrombotic gene mutations have been related to vascular disorders and sudden hearing loss. We assessed the prothrombotic risk in 10 patients with sudden sensorineural hearing loss who had previously experienced cardiovascular events to support its vascular pathogenesis. Methods: Ten patients underwent hematologic tests (MTHFR C677T/A1298C, prothrombin G20210A, platelet GlyIIIaA1/A2, and V Leiden G1691A genotyping; fibrinogenemia; cholesterolemia; homocysteinemia; folatemia). The results were compared with those of 100 previously investigated patients with sudden hearing loss alone and those of 200 healthy controls. DNA was isolated from peripheral blood leukocytes, and the gene mutations were investigated by polymerase chain reaction and a LightCycler DNA analyzer. Results: Two patients had 2 mutant alleles, 6 had 3, and 2 had 4. The mean homocysteine, cholesterol, and fibrinogen levels were above the upper limit of normal; the mean folate levels were slightly above the lower limit of normal. Multiple mutations were more frequent in the patient group than in the previously analyzed patients and healthy controls. Conclusions: The association between inherited and acquired prothrombotic factors in patients with sudden sensorineural hearing loss and thrombotic diseases in other sites suggests that a multifactorial mechanism may underlie microvascular cochlear impairment. Hematologic investigation, including MTHFR, prothrombin, platelet, and V Leiden genotyping, may help to detect patients at potential risk of recurrent hearing loss and multiple microvascular diseases, and could be usefully performed in otherwise idiopathic sudden sensorineural hearing loss.

Differences in size at birth are determined by differences in growth velocity during early prenatal life.

Physiologic interindividual differences in neonatal size are traditionally thought of as determined by differences in fetal growth occurring only in the second half of pregnancy. Whether possible differences in early intrauterine growth velocity are the effect of random growth fluctuations or may affect size at birth is still debated. This article aims at evaluating to what extent differences in neonatal size are accounted for by differences in fetal growth velocity. We analyzed the fetal growth of 130 healthy singletons for whom head (HC) and abdomen (AC) circumferences and femur diaphysis length (FDL) longitudinal profiles were available, together with the measures of weight (BW), length (BL), and head circumference (BHC) at birth. Individual profiles were fitted with ad-hoc models. Neonatal traits were transformed into standard deviation scores (SDS). Neonates in the upper third of BW-SDS distribution (3618 ± 43 g, mean ± SEM) had, at 22 wk of gestational age, AC growth velocity higher by 0.55 ± 0.10 mm/wk than those in the lower third (2902 ± 36 g). Neonates in the upper third of BL-SDS distribution (51.7 ± 0.21 cm) had, at 20 wk, FDL growth velocity higher by 0.11 ± 0.05 mm/wk than those in the lower third (48.2 ± 0.18 cm). Neonates in the upper third of BHC-SDS distribution (35.7 ± 0.13 cm) had, at 18 wk, HC growth velocity higher by 0.57 ± 0.20 mm/wk than those in the lower third (33.3 ± 0.11 cm). The differences in growth velocity remain constant throughout the second and third trimester for AC, and tend to vanish in the third trimester for HC and FDL. The differences in fetal growth velocity, which in our study were observed as early as mo 4, suggest that the genetic component plays an important role in fetal growth and is precociously expressed.

Classification criteria and distinction between migraine and tension-type headache in children

The International Headache Society (IHS) classification system (1988) was developed primarily for headache disorders in adults and its validity for paediatric age is currently under discussion; in 1995 Seshia et al. proposed a revision of the criteria for migraine without aura to make diagnostic criteria more applicable to children. The purposes of the current study were to: (1) compare the IHS classification with the Seshia proposal, (2) compare the children affected by migraine without aura (MO) with the children affected by tension headache (TH) as defined by Seshia, for characteristics which are not included in the classification. The patients are a series of 320 children (mean age 9.9, SD 2.6 years; 144 males, 176 females) with recurrent or chronic headaches referred to a headache clinic in Milan, Italy. Using the Seshia criteria instead of the IHS criteria a higher number of children were included in the MO category: bilateral pain and family history of migraine were the most important factors which allowed a shift of children into this category. However, with the Seshia classification there was no reduction in the number of unclassifiable children. The reason why some children could not be classified was a short duration of attacks; the majority of unclassifiable children were 6 years old or less. No relevant difference was found between children with MO and children with TH for the following variables: occurrence of attacks in the afternoon or evening after school, reduction of attacks during school holidays, full‐time schooling, after‐school activities on school days, disordered daily life. On the contrary children with MO when compared with those with TH showed a higher number of precipitating factors and for the following factors a significant difference was found: exposure to TV or a computer, sleep deficiency, and strong emotions. Furthermore, children with MO showed a greater severity of attacks.

Reproducibility of the WHO histological criteria for the diagnosis of Philadelphia chromosome-negative myeloproliferative neoplasms

This study, performed on behalf of the Italian Registry of Thrombocythaemias (Registro Italiano Trombocitemie), aimed to test the inter-observer reproducibility of the histological parameters proposed by the WHO classification for the diagnosis of the Philadelphia chromosome-negative myeloproliferative neoplasms. A series of 103 bone marrow biopsy samples of Philadelphia chromosome-negative myeloproliferative neoplasms consecutively collected in 2004 were classified according to the WHO criteria as follows: essential thrombocythaemia (n=34), primary myelofibrosis (n=44) and polycythaemia vera (n=25). Two independent groups of pathologists reviewed the bone marrow biopsies. The first group was asked to reach a collegial ‘consensus’ diagnosis. The second group reviewed individually all the cases to recognize the main morphological parameters indicated by the WHO classification and report their results in a database. They were subsequently instructed to individually build a ‘personal’ diagnosis of myeloproliferative neoplasms subtype just assembling the parameters collected in the database. Our results indicate that high levels of agreement (≥70%) have been reached for about all of the morphological features. Moreover, among the 18 evaluated histological features, 11 resulted statistically more useful for the differential diagnosis among the different Philadelphia chromosome-negative myeloproliferative neoplasms. Finally, we found a high percentage of agreement (76%) between the ‘personal’ and ‘consensus’ diagnosis (Cohen’s kappa statistic >0.40). In conclusion, our results support the use of the histological criteria proposed by the WHO classification for the Philadelphia chromosome-negative myeloproliferative neoplasms to ensure a more precise and early diagnosis for these patients.

Diagnostic criteria for migraine and psychogenic headache in children

The headache histories obtained from 214 children were analysed by computer to see whether it was possible to identify and classify migraine, and to distinguish children with psychogenic headache. During headache attacks, most children had no or very few associated symptoms. For classification, 175 patients were divided into four homogeneous groups; the remaining 39 could not be grouped. An overlap between the different groups was found. Psychogenic headache emerged as a clearly definable syndrome, characterised by psychological problems and daily headache for a period of at least one month (10 patients). When the 214 patients were grouped according to the classification of the Headache Classification Committee of the International Headache Society, distinguishing those children with psychogenic headache was no longer possible.

What Is the Incidence of Cystic Fibrosis in Italy? Data from the National Registry (1988-2001)

The mean incidence of cystic fibrosis (CF) among North Americans of European ancestry is 1 in 2,500. Studies carried out in different geographic areas report incidence values ranging from 1 in 1,800 to 1 in 8,500, suggesting that incidence has to be assessed specifically for any population. This paper represents the first attempt to evaluate the incidence of CF in Italy and its time-related changes following the discovery of the gene in 1989. Data recorded in the Italian National CF Registry for CF subjects born between 1988 and 2001 were evaluated. All CF patients (1,742) were diagnosed by 1 of 18 specialized CF centers and 7 support services (1-3 in each region) according to the consensus statement on CF diagnosis. The average CF incidence in Italy was found to be 1 in 4,238, which is lower than the values reported elsewhere. The incidence remained nearly constant from 1988 to 2001, although the percentage of subjects screened for CF increased by 44%. Molecular analysis (available for more than 80% of the patients) indicated that the most common mutation (DF508) in Italy occurs in only 49% of CF chromosomes; the other mutations differ from region to region. These differences appeared to be mildly related to differences in reason for diagnosis and type of mutation. Italian registry data supply an incidence of CF lower than that reported for populations of European descent. Part of this difference is probably due to the higher allelic heterogeneity of the CFTR gene in Italy with respect to Northern Europe or the U.S. population of European descent.

Longitudinal distance standards of fetal growth

Background. Most ultrasonographic fetal growth norms are derived from cross‐sectional data or from longitudinal data treated as coming from cross‐sectional studies, although only longitudinal models may detect particular aspects of fetal growth shape, such as peak of growth velocity.