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Abstract The question addressed by the study Good biological indicators capable of predicting chronic obstructive pulmonary disease (COPD) phenotypes and clinical trajectories are lacking. Because nuclear and mitochondrial genomes are damaged and released by cigarette smoke exposure, plasma cell-free mitochondrial and nuclear DNA (cf-mtDNA and cf-nDNA) levels could potentially integrate disease physiology and clinical phenotypes in COPD. This study aimed to determine whether plasma cf-mtDNA and cf-nDNA levels are associated with COPD disease severity, exacerbations, and mortality risk. Materials and methods We quantified mtDNA and nDNA copy numbers in plasma from participants enrolled in the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE, n = 2,702) study and determined associations with relevant clinical parameters. Results Of the 2,128 participants with COPD, 65% were male and the median age was 64 (interquartile range, 59–69) years. During the baseline visit, cf-mtDNA levels positively correlated with future exacerbation rates in subjects with mild/moderate and severe disease (Global Initiative for Obstructive Lung Disease [GOLD] I/II and III, respectively) or with high eosinophil count (≥ 300). cf-nDNA positively associated with an increased mortality risk (hazard ratio, 1.33 [95% confidence interval, 1.01–1.74] per each natural log of cf-nDNA copy number). Additional analysis revealed that individuals with low cf-mtDNA and high cf-nDNA abundance further increased the mortality risk (hazard ratio, 1.62 [95% confidence interval, 1.16–2.25] per each natural log of cf-nDNA copy number). Answer to the question Plasma cf-mtDNA and cf-nDNA, when integrated into quantitative clinical measurements, may aid in improving COPD severity and progression assessment.

This work introduces a real-time intention decoding algorithm grounded in muscle synergies (Syn-ID). The algorithm detects the electromyographic (EMG) onset and infers the direction of the movement during reaching tasks to control a powered shoulder–elbow exoskeleton. Features related to muscle synergies are used in a Gaussian Mixture Model and probability accumulation-based logic to infer the user’s movement direction. The performance of the algorithm was verified by a feasibility study including eight healthy participants. The experiments comprised a transparent session, during which the exoskeleton did not provide any assistance, and an assistive session in which the Syn-ID strategy was employed. Participants were asked to reach eight targets equally spaced on a circumference of 25 cm radius (adjusted chance level: 18.1%). The results showed an average accuracy of 48.7% after 0.6 s from the EMG onset. Most of the confusion of the estimate was found along directions adjacent to the actual one (type 1 error: 33.4%). Effects of the assistance were observed in a statistically significant reduction in the activation of Posterior Deltoid and Triceps Brachii. The final positions of the movements during the assistive session were on average 1.42 cm far from the expected ones, both when the directions were estimated correctly and when type 1 errors occurred. Therefore, combining accurate estimates with type 1 errors, we computed a modified accuracy of 82.10±6.34%. Results were benchmarked with respect to a purely kinematics-based approach. The Syn-ID showed better performance in the first portion of the movement (0.14 s after EMG onset).

Abstract Purpose Extracorporeal membrane oxygenation (ECMO) has been increasingly used in the last years to provide hemodynamic and respiratory support in critically ill patients. In this scenario, prognostic scores remain essential to choose which patients should initiate ECMO. This systematic review aims to assess the current landscape and inform subsequent efforts in the development of risk prediction tools for ECMO. Methods PubMed, CINAHL, Embase, MEDLINE and Scopus were consulted. Articles between Jan 2011 and Feb 2022, including adults undergoing ECMO reporting a newly developed and validated predictive model for mortality, were included. Studies based on animal models, systematic reviews, case reports and conference abstracts were excluded. Data extraction aimed to capture study characteristics, risk model characteristics and model performance. The risk of bias was evaluated through the prediction model risk-of-bias assessment tool (PROBAST). The protocol has been registered in Open Science Framework ( https://osf.io/fevw5 ). Results Twenty-six prognostic scores for in-hospital mortality were identified, with a study size ranging from 60 to 4557 patients. The most common candidate variables were age, lactate concentration, creatinine concentration, bilirubin concentration and days in mechanical ventilation prior to ECMO. Five out of 16 venous-arterial (VA)-ECMO scores and 3 out of 9 veno-venous (VV)-ECMO scores had been validated externally. Additionally, one score was developed for both VA and VV populations. No score was judged at low risk of bias. Conclusion Most models have not been validated externally and apply after ECMO initiation; thus, some uncertainty whether ECMO should be initiated still remains. It has yet to be determined whether and to what extent a new methodological perspective may enhance the performance of predictive models for ECMO, with the ultimate goal to implement a model that positively influences patient outcomes.

Background: There is still no consensus on perioperative pain control techniques in patients undergoing laparoscopic surgery; protocols of conventional therapy can be improved by the use of perioperative anaesthesiologic techniques, such as epidural or loco-regional analgesic administration as transversus abdominis plane (TAP) block. The aim of this evaluation was to investigate the role of laparoscopic-assisted TAP block during repair of diastasis recti associated with primary midline hernias in term of post-operative pain relief. Materials and Methods: This was a retrospective evaluation of a prospectively maintained database including patients undergoing laparoscopic repair of diastasis recti associated with primary ventral hernia. Patients were divided into two groups: Group A patients (n = 34) received laparoscopic-assisted bilateral TAP-block of 7.5 mg/ml ropivacaine for each side and Group B patients (n = 29) received conventional post-operative therapy. All patients received 24 h infusion of 20 mg morphine; pain was checked at 6, 24 and 48 h after surgery by numeric rating scale (NRS) score. A rescue analgesia by was given if NRS score was >4 or on patient request. Results: No differences in operative time, complications and post-operative stay, no complications related to TAP-block technique were found. Post-operative pain scores (determined by NRS) were found to be significantly different between groups. Group A patients showed a significant reduction in NRS score at 6, 24 and 48 h (P < 0.005) and in the number of patients requiring further analgesic drugs administration (P < 0.005) compared to Group B patients. Conclusions: Laparoscopic-guided TAP-block can be considered safe and effective in the management of post-operative pain and in the reduction of analgesic need in patients undergoing laparoscopic repair of diastasis recti and ventral hernias. The non-randomised nature of the study and the lack of a consistent series of patients require further evaluations.

A reaction in anhydrous toluene between the formally unsaturated fragment [Ln(hfac)3] (Ln3+ = Eu3+, Gd3+ and Er3+; Hhfac = hexafluoroacetylacetone) and [Al(qNO)3] (HqNO = 8-hydroxyquinoline N-oxide), here prepared for the first time from [Al(OtBu)3] and HqNO, affords the dinuclear heterometallic compounds [Ln(hfac)3Al(qNO)3] (Ln3+ = Eu3+, Gd3+ and Er3+) in high yields. The molecular structures of these new compounds revealed a dinuclear species with three phenolic oxygen atoms bridging the two metal atoms. While the europium and gadolinium complexes show the coordination number (CN) 9 for the lanthanide centre, in the complex featuring the smaller erbium ion, only two oxygens bridge the two metal atoms for a resulting CN of 8. The reaction of [Eu(hfac)3] with [Alq3] (Hq = 8-hydroxyquinoline) in the same conditions yields a heterometallic product of composition [Eu(hfac)3Alq3]. A recrystallization attempt from hot heptane in air produced single crystals of two different morphologies and compositions: [Eu2(hfac)6Al2q4(OH)2] and [Eu2(hfac)6(µ-Hq)2]. The latter compound can be directly prepared from [Eu(hfac)3] and Hq at room temperature. Quantum mechanical calculations confirm (i) the higher stability of [Eu(hfac)3Al(qNO)3] vs. the corresponding [Eu(hfac)3Alq3] and (ii) the preference of the Er complexes for the CN 8, justifying the different behaviour in terms of the Lewis acidity of the metal centre.

Gene and genome comparison represent an invaluable tool to identify evolutionarily conserved sequences with possible functional significance. In this work, we have analyzed orthologous genes encoding subunits and assembly factors of the V-ATPase complex, an important enzymatic complex of the vacuolar and lysosomal compartments of the eukaryotic cell with storage and recycling functions, respectively, as well as the main pump in the plasma membrane that energizes the epithelial transport in insects. This study involves 70 insect species belonging to eight insect orders. We highlighted the conservation of a short sequence in the genes encoding subunits of the V-ATPase complex and their assembly factors analyzed with respect to their exon-intron organization of those genes. This study offers the possibility to study ultra-conserved regulatory elements under an evolutionary perspective, with the aim of expanding our knowledge on the regulation of complex gene networks at the basis of organellar biogenesis and cellular organization.

An innovative strengthening system for dapped-end beams is studied numerically and experimentally in this paper. The system is developed for the half-joint regions of bridge beams also commonly called “gerber saddles”, but it can be adapted to different scenarios. The strengthening system consists of two steel plates that are clamped on both sides of the webs of the beams by means of bolts. The purpose of the system is to transfer the highest possible amount of shear from the concrete webs to the steel plate elements reducing the resistance demand of the concrete half joint. Shear is transferred by friction from concrete to steel plates. The system is designed to be applied on existing bridges without heavy work interesting the carriageway, therefore reducing the interference with the traffic. Some interesting considerations emerge from the study, including the influence of the flange web connection on the structural behavior and the possible presence of brittle failure mechanisms that are difficult to model numerically using f.e.m. simulations.

Background: Intra- and juxta-articular osteoid osteomas are rare, representing less than 10% of all osteomas. Compared to the classic diaphyseal or metaphyseal site of long bones, they often have an atypical onset, a longest diagnostic delay, and frequent initial misdiagnoses, with pictures that can mimic inflammatory monoarthritis. We aimed to describe a case series, and to provide a literature review of this uncommon and misleading tumor location. Methods: We performed a retrospective analysis of patients referred to three pediatric rheumatology centers, with a final diagnosis of articular osteoid osteoma. A review of the literature was additionally conducted. Results: We included 10 patients with a mean age of 14 years. All patients with unusual sites (olecranon fossa, lumbar vertebra, distal phalanx of the toe, fibula) had a misdiagnosis, and cases with initial suspicion of monoarthritis had the longest diagnostic delay, up to 24 months. The literature review confirms the significant risk of misdiagnosis, and an average time from symptom onset to diagnosis ranging from 0.4 to 1.8 years. Conclusions: Articular osteoid osteoma may mimic arthritis, especially in adolescence. Knowledge of the atypical forms of presentation, and of the clinical and radiological pitfalls, reduces the risk of diagnostic error.

The safety assessment of existing structures in areas with a relevant seismic hazard is one of the major topics for engineers since many existing reinforced concrete structures have been realized disregarding seismic design with minimal details with respect to present practice. In this context, seismic assessment is a primary issue in order to identify the best retrofitting solution with the aim of enhancing the efficiency of existing buildings. In recent years, with the aim to enhance the seismic behavior of reinforced concrete (RC) structures (with particular care to existing ones), the system of seismic isolation adopting friction pendulum (FPS) devices proved to be among the most diffuse and effective solutions. The purpose of this paper is to explore the effectiveness of the refurbishment using FPS with single concavity devices on the performance of one irregular existing RC building placed in a highly seismic area of central Italy. First, the geometric and material characteristics of the building have been determined within the approach based on the “knowledge levels”. Second, a suitable numerical model based on a fiber-modeling approach has been established using SAP2000, including relevant mechanical non-linearities. Then, a set of 21 natural seismic inputs, inclusive of 3 accelerations over vertical and horizontal directions, was adopted with the aim of performing non-linear (NL) dynamic simulations. The NL dynamic simulations have been performed considering the structural system, both inclusive and not inclusive of the FPS isolator devices. The influence of the actual distribution of infill masonry panels on the overall behavior of the structure has also been evaluated in both of the cases mentioned above. Finally, the outcomes deriving from the NL dynamic simulations were helpful in assessing the advantages of the intervention of retrofitting to improve the seismic performance of the building, highlighting the influence of masonry infills.

Patients are the most important actors in clinical research. Therefore, patient preference information (PPI) could support the decision-making process, being indisputable for research value, quality, and integrity. However, there is a lack of clear guidance or consensus on the search for preference studies. In this blueprint, an openly available and regularly updated patient preference management system for an integrated database (PPMSDB) that contains the minimal set of data sufficient to provide detailed information for each study (the so-called evidence tables in systematic reviews) and a high-level overview of the findings of a review (summary tables) is described. These tables could help determine which studies, if any, are eligible for quantitative synthesis. Finally, a web platform would provide a graphical and user-friendly interface. On the other hand, a set of APIs (application programming interfaces) would also be developed and provided. The PPMSDB, aims to collect preference measures, characteristics, and meta-data, and allow researchers to obtain a quick overview of a research field, use the latest evidence, and identify research gaps. In conjunction with proper statistical analysis of quantitative preference measures, these aspects can facilitate formal evidence-based decisions and adequate consideration when conducting a structured decision-making process. Our objective is to outline the conceptual infrastructure necessary to build and maintain a successful network that can monitor the currentness and validity of evidence.

Cigarette smoke (CS) is the most common risk factor for chronic obstructive pulmonary disease (COPD). The present study aimed to elucidate whether mtDNA is released upon CS exposure and is detected in the plasma of former smokers affected by COPD as a possible consequence of airway damage. We measured cell-free mtDNA (cf-mtDNA) and nuclear DNA (cf-nDNA) in COPD patient plasma and mouse serum with CS-induced emphysema. The plasma of patients with COPD and serum of mice with CS-induced emphysema showed increased cf-mtDNA levels. In cell culture, exposure to a sublethal dose of CSE decreased mitochondrial membrane potential, increased oxidative stress, dysregulated mitochondrial dynamics, and triggered mtDNA release in extracellular vesicles (EVs). Mitochondrial DNA release into EVs occurred concomitantly with increased expression of markers that associate with DNA damage responses, including DNase III, DNA-sensing receptors (cGAS and NLRP3), proinflammatory cytokines (IL-1β, IL-6, IL-8, IL-18, and CXCL2), and markers of senescence (p16 and p21); the majority of the responses are also triggered by cytosolic DNA delivery in vitro. Exposure to a lethal CSE dose preferentially induced mtDNA and nDNA release in the cell debris. Collectively, the results of this study associate markers of mitochondrial stress, inflammation, and senescence with mtDNA release induced by CSE exposure. Because high cf-mtDNA is detected in the plasma of COPD patients and serum of mice with emphysema, our findings support the future study of cf-mtDNA as a marker of mitochondrial stress in response to CS exposure and COPD pathology.

In many countries around the world a huge number of existing reinforced concrete (RC) structures have been realized without account for seismic detailing, even if they are located in areas subjected to high seismicity. In this context, several passive seismic protection techniques have been developed and applied to retrofit these structures such as, for an example, seismic isolation. The aim of this work is to characterize in probabilistic terms the seismic performance of a framed RC building retrofitted by means of sliding friction pendulum (FPS) devices. Specifically, the response of an existing RC building located in a high seismicity area in Italy is investigated. After the description of the main available information about the structure, a non-linear numerical model has been defined by means of fiber-elements approach. Then, non-linear dynamic analyses considering multiple recorded ground motions with the three accelerometric components have been carried out to assess the seismic response of the building with and without the retrofitting intervention composed of FPS isolators. Finally, the results are processed to achieve a probabilistic assessment of the seismic performance of the retrofitting intervention.

BACKGROUND:The primary objective of this study was to retrospectively compare short-term outcomes of intracorporeal versus extracorporeal anastomosis for minimally invasive laparoscopic and robotic-assisted right colectomies for benign and malignant disease. Recent studies suggest potential short-term outcomes advantages for the intracorporeal anastomosis technique. METHODS:This is a multicenter retrospective propensity score-matched comparison of intracorporeal and extracorporeal anastomosis techniques for laparoscopic and robotic-assisted right colectomy between January 11, 2010, and July 21, 2016. RESULTS:After propensity score-matching, there were a total of 1029 minimal invasive surgery cases for analysis-379 right colectomies (335 robotic-assisted and 44 laparoscopic) done with an intracorporeal anastomosis and 650 right colectomies (253 robotic-assisted and 397 laparoscopic) done with an extracorporeal anastomosis. There were no significant differences in any preoperative patient characteristics between groups. The minimally invasive intracorporeal anastomosis group had significantly longer operative times (p

Mitochondrial disorders associated with genetic defects of the ATP synthase are among the most deleterious diseases of the neuromuscular system that primarily manifest in newborns. Nevertheless, the number of established animal models for the elucidation of the molecular mechanisms behind such pathologies is limited. In this paper, we target the Drosophila melanogaster gene encoding for the ATP synthase subunit c, ATPsynC, in order to create a fruit fly model for investigating defects in mitochondrial bioenergetics and to better understand the comprehensive pathological spectrum associated with mitochondrial ATP synthase dysfunctions. Using P-element and EMS mutagenesis, we isolated a set of mutations showing a wide range of effects, from larval lethality to complex pleiotropic phenotypes encompassing developmental delay, early adult lethality, hypoactivity, sterility, hypofertility, aberrant male courtship behavior, locomotor defects and aberrant gonadogenesis. ATPsynC mutations impair ATP synthesis and mitochondrial morphology, and represent a powerful toolkit for the screening of genetic modifiers that can lead to potential therapeutic solutions. Furthermore, the molecular characterization of ATPsynC mutations allowed us to better understand the genetics of the ATPsynC locus and to define three broad pathological consequences of mutations affecting the mitochondrial ATP synthase functionality in Drosophila: i) pre-adult lethality; ii) multi-trait pathology accompanied by early adult lethality; iii) multi-trait adult pathology. We finally predict plausible parallelisms with genetic defects of mitochondrial ATP synthase in humans.

Purpose: A prospective study was conducted to evaluate signal changes in the dentate nucleus, globus pallidus, pons, and thalamus (normalized to the deep cerebellum white matter) in T1-weighted magnetic resonance (MR) images after serial injections of gadobutrol in patients with thalassemia without neurological lesions., Methods: In this study three groups were scanned at both 1.5 T and 3 T: 15 thalassemia patients transfused and chelated with ≥4 gadobutrol administrations at a high dose (0.2 mmol/kg per scan) for late gadolinium enhancement (LGE) cardiovascular MR, 8 thalassemia patients and 13 healthy subjects who had never received gadolinium-based contrast agents (GBCA)., Results: Signal intensity (SI) ratios at 1.5 T in all regions were comparable among the three groups and were not correlated with the number of gadobutrol administrations. In healthy subjects SI ratios were significantly different among the 4 regions, being higher in the pallidus. The SI ratios at 1.5 T were significantly higher and not correlated with SI ratios at 3 T or with iron overload in the same regions assessed by the T2* technique., Conclusion: This article describes the lack of increased SI in T1-weighted MR images after repeated administration of gadobutrol for cardiovascular MR studies in a high-risk population (high dose per scan, iron overload that can facilitate the transmetalation of gadolinium) scanned at 3 T and 1.5 T.

Introduction: SIOPEN INES protocol yielded excellent 5-year survival rates for MYCN-non-amplified metastatic neuroblastoma. Patients deemed ineligible due to lack or delay of MYCN status or late registration were treated, but not included in the study. Our goal was to analyse survival at 10 years among the whole population., Materials and Methods: Italian and Spanish metastatic INES patients' data are reported. SPSS 20.0 was used for statistical analysis., Results: Among 98 infants, 27 had events and 19 died, while 79 were disease free. Five- and 10-year event-free survival (EFS) were 73 and 70 %, and overall survival (OS) was 81 and 74 %, respectively. MYCN status was significant for EFS, but not for OS in multivariate analysis., Conclusions: The survival rates of patients who complied with all the inclusion criteria for INES trials are higher compared to those that included also not registered patients. Five-year EFS and OS for INES 99.2 were 87.8 and 95.7 %, while our stage 4s population obtained 78 and 87 %. Concerning 99.3, 5-year EFS and OS were 86.7 and 95.6 %, while for stage 4 we registered 61 and 68 %. MYCN amplification had a strong impact on prognosis and therefore we consider it unacceptable that many patients were not studied for MYCN and probably inadequately treated. Ten-year survival rates were shown to decrease: EFS from 73 to 70 % and OS from 81 to 74 %, indicating a risk of late events, particularly in stage 4s. Population-based registries like European ENCCA WP 11-task 11 will possibly clarify these data.

Background and Aims: Branchial abnormalities occur when there is disturbance in the maturation of the branchial apparatus during fetal development. Branchial anomalies are congenital lesions usually present in childhood, even if they can be diagnosed later for enlargement or infection. A correct diagnosis will lead to proper management: complete surgical excision is the treatment of choice. The purpose of this article is to present clinical features, diagnostic methods and surgical treatment of branchial anomalies in childhood, based on a series of 50 patients., Methods: We conducted a retrospective analysis of a total of 50 pediatric patients operated from June 2005 to June 2014 for the presence of branchial cleft anomalies., Results: 27 cases (54 %) presented a second branchial cleft fistula and 11 cases (22 %) a second branchial cleft cyst and one case (2 %) presented both cyst and sinus of the second branchial cleft; four cases (8 %) presented first branchial cleft cyst whereas four cases (8 %) a first branchial cleft sinus and two cases (4 %) a first branchial cleft fistula; one case (2 %) presented a piriform sinus fistula (third branchial cleft). None of our patients presented anomalies of the fourth branchial cleft. All patients underwent surgical treatment and lesions have been removed by excision or fistulectomy. No post-surgical complication occurred. The rate of recurrence was 4 %., Conclusions: Pre-operative diagnosis supplies important information to the surgeon for a proper therapy: a complete excision of the lesion without inflammatory signs is essential to avoid re-intervention and to achieve a good outcome.

Henoch Schonlein purpura (HSP) is a small-sized blood vessels vasculitis, resulting from immunoglobulin A-mediated inflammation. It is the most common acute systemic vasculitis in childhood and mainly affects skin, gastrointestinal tract, joints and kidney. Skin lesions, usually presenting as erythematous maculopapules, petechiae, and purpura, often involve lower extremities and buttocks, but may also extend to the upper extremities, face and trunk. Conversely to adults, hemorrhagic bullous evolution has been seldom described in childhood. The pressure is likely a factor into the pathogenesis of bullae. We report on three new pediatric cases of HSP with hemorrhagic bullous skin lesions, and a review of the literature. Bullous evolution represents an unusual, but well-recognized cutaneous manifestation that may be a source of diagnostic dilemma, but does not seem to have any prognostic value in the outcome of HSP.

A 12-year-old girl with latent Mycobacterium tuberculosis infection and currently receiving prophylaxis with isoniazid presented with nausea, vomiting and increasing frontal headache. Toxicity to the anti-tuberculosis drug was suspected, but her symptoms persisted after isoniazid withdrawal. A computed tomography (CT) scan showed a brain mass to be present.

There are no data concerning the significance of allergen specific nasal challenge to latex (ASNCL) in the pediatric population and the effect of mometasone furoate nasal spray (MFNS), topic corticosteroid exerting a potent anti-inflammatory activity in children with latex allergic rhinitis. The aims of this study are: to investigate the clinical and immune pathological effects of ASNCL in children with latex allergy; to study the effects of MFNS pre-medication on the clinical and immune pathological effects of ASNCL in children with latex allergy. Thirteen children: 6 male and 7 female, mean (SD) age 9.6 (2.9) years, with latex allergy and seven children: 3 male and 4 female, mean (SD) age 9.9 (3.8) years, without latex allergy underwent ASNCL. Nasal symptoms were recorded, nasal lavage fluid was collected to measure tryptase, eosinophil cationic protein (ECP), interleukin-5, interferon-gamma levels, and spirometric test was performed for each patient without or with premedication with MFNS. ASNCL induced a clinical allergic response and increased tryptase levels only in children with latex allergy. No serious adverse events occurred after ASNCL. MFNS premedication reduced both tryptase and ECP levels only in children with latex allergy. ASNCL is a simple, reliable and useful tool to make or confirm the diagnosis of nasal symptoms due to latex; it allows us to study both clinical symptoms and local immunological changes. MFNS premedication before an ASNCL may prevent some immunological responses induced by ASNCL without clinical allergic modifications.

To analyse short and long-latency (SEPs) recorded by chronically stereotactically electrodes implanted in SI area of two epileptic patients. Two drug-resistant epileptic patients (2 females, 38 and 15 years, respectively) suffering from left temporal and right frontal epilepsy respectively, were investigated by an electrode-chronically implanted in SI area. Short and long latency somatosensory evoked potentials were recorded by depth electrodes 10 days after implantation. This is the first study to describe a depth N36 response by an intracerebral recording electrode in the SI area, probably generated by a radially oriented generator, located in area 1. Furthermore, we confirmed a role of SI in the genesis of N60 component. Finally, our present data suggest that the SI area is still active at 120 ms after the stimulus, since in one patient (no. 2) we identified a N120 potential, reaching its maximal amplitude at the same depth as the N20 response.

Peritoneal and extracorporeal dialysis are used to treat newborns affected by inborn errors of metabolism to minimize the effects of the acute accumulation of neurotoxic metabolites that can produce irreversible and severe neurological damage and even death. In recent papers, extracorporeal dialysis has been described as more effective than peritoneal dialysis in improving the prognosis in newborns with inborn errors of metabolism and hyperammonemia. However, it appears that the outcome is primarily related to the duration of neonatal hyperammonemic coma. Here we report seven newborns with hyperammonemia caused by inborn errors of metabolism (five with organic acidemias, two with urea-cycle disorders). They received dietetic and pharmacological treatment as well as peritoneal dialysis. Four of the five patients with organic acidemia survived with and without mild neurological impairment (follow-up 3.5-10 years). One died from bacterial sepsis after peritoneal dialysis was discontinued and the peritoneal catheter was removed. One of the two patients affected by urea-cycle disorders, a boy, died during the neonatal period, and the other, a girl, died at the age of 13 months due to severe neurological damage. Our results demonstrate that peritoneal dialysis may still be an effective treatment for neonatal hyperammonemia caused by inborn errors of metabolism. Furthermore, peritoneal dialysis can be administered quickly and easily in all settings, clearly an advantage when fast intervention is so crucial.

Over the last 2 decades, the survival rate of infants born at < or = 25 weeks of gestation has increased; however, significant morbidity and disability persist. The commitment for their care gives rise to a variety of complex medical, social, and ethical aspects. Decision-making is a crucial issue that involves the infant, the family, health care providers, and society. In a review of the existing guidelines, we investigated the different approaches in the care of extremely preterm births in various countries. We found that many scientific societies and professional organizations have issued guidelines that address the recommendations for the care of these fetuses/neonates although to varying degrees. In this article we compare different approaches and assess the scientific grounds of the specific recommendations. With current standards, intensive care is generally considered justifiable at > or = 25 weeks, compassionate care at < or = 22 weeks, and an individual approach at 23 to 24 weeks, consistent with the parents' wishes and the infant's clinical conditions at birth.

Background: A retrospective study was conducted to provide epidemiological data on hospitalization for complicated and uncomplicated chickenpox in a pediatric population., Methods: The study analyzed hospitalization cases for chickenpox, among all the 31 Tuscan hospitals, during the period 1997-2003., Results: Globally, 650 cases were recorded (306 = 47.07% for uncomplicated and 344 = 52.92% for complicated chickenpox). Total hospitalization rate was 22.66 per 100,000 living Tuscan children and 11.52 per 1,000 notified chickenpox cases. Hospitalization rates for complicated chickenpox were 12.00 per 100,000 living children and 6.09 per 1,000 notified cases. Notably, significantly increased hospitalization rates for complicated chickenpox were evidenced over years (p = 0.011 per 100,000 living children and p = 0.001 per 1,000 notified cases), due to the increased proportion of neurological (p = 0.043 per 100,000 living children and p = 0.025 per 1,000 notified cases) and respiratory (p = 0.021 per 100,000 living children and p = 0.008 per 1,000 notified cases) complications, whereas hospitalization rates for other complications as well as for uncomplicated chickenpox remained constant (p = 0.25 per 100,000 living children and p = 0.09 per 1,000 notified cases)., Conclusions: Chickenpox complications, requiring hospitalization, occurred at a substantial rate in our pediatric population. In particular, increasing hospitalization rates for neurological and respiratory complications were evidenced over the study period. Our epidemiological data may provide additional information while planning a vaccination strategy for Italy.

Background: The introduction of HAART has decreased mortality and progression to AIDS in perinatally HIV-1-infected children, but information on modification of the rate of specific clinical events is limited., Method: An observational population study on changes in HIV-1-related morbidity was conducted on 1402 perinatally HIV-1-infected children enrolled in the Italian Register for HIV Infection in Children and prospectively followed in the pre-HAART (1985-1995) and post-HAART periods (1996-2000, and 2001-2005). Of this group, 773 children (55.1%) were followed from birth. Median observation time was 8.58 years (interquartile range, 3.71-13.72)., Results: Overall, 666 (47.5%) children developed AIDS and 420 (29.9%) died. Improved survival over time was evidenced at Kaplan-Meier analysis (P < 0.0001). Poisson regression analysis indicated that Centers for Disease Control and Prevention class B and C clinical event rates and most of the HIV-1-related organ complication rates significantly decreased starting from 1996-2000. Significant reductions in rates of cancer and opportunistic infections were evidenced after 2000. Nevertheless, opportunistic infections still occurred at high rates (6.09/100 person-years) in 2001-2005, with high rate of bacterial infections (3.55/100 person-years), particularly pneumonia (1.66/100 person-years), in this period. CD4 cell percentage was > 15% in 58.5% children with pneumonia., Conclusions: Progressive reductions of both mortality and rates of class B and C clinical events, including organ complications, were evidenced in the HAART era. Nevertheless, severe bacterial infections, particularly pneumonia, still occurred at considerable high rates, even in the absence of a severe CD4 cell depletion.

Background: The most frequent noncutaneous site of involvement of chickenpox is the central nervous system (CNS) and complications include cerebellar ataxia, encephalitis, and meningitis., Objectives: We have recently observed an unusually high number of children with chickenpox CNS complications in our university children's hospital. A study to evaluate the incidence of these complications over time in children living in Tuscany was carried out., Study Design: We evaluated all cases of chickenpox and chickenpox complications leading to hospitalization in children aged 1 month-14 years reported to the Tuscany public health centre between 1997 and 2004. The International Classification of Disease Ninth Revision-CM hospital discharge diagnostic codes and medical records were used., Results: The incidence (95% confidence interval) of CNS complications/1000 chickenpox cases was stable between 1997 and 2001 [1997: 0.80 (0.29-1.74); 1998: 0.73 (0.29-1.50); 1999: 0.67 (0.25-1.46); 2000: 0.56 (0.15-1.44); 2001: 0.59 (0.16-1.50)] but increased significantly (chi(2) for trend: 9.401; p=.0021) in 2002 [1.56 (0.83-2.66)], in 2003 [1.73 (0.95-2.90)] and in 2004 [1.51 (0.74-2.27)]. Non-CNS complications remained stable over time., Conclusions: Possible factors biasing the result were taken into account. Reasons of increased CNS complications remain unknown, but the possible emergence of a particularly neurotropic strain of varicella-zoster virus should be further investigated.

Background: Subcortical band heterotopia (SBH, or double cortex syndrome) is a neuronal migration disorder consisting of heterotopic bands of gray matter located between the cortex and the ventricular surface, with or without concomitant pachygyria. Most cases show diffuse or anteriorly predominant (A>P) migration abnormality. All familial and 53% to 84% of sporadic cases with diffuse or A>P SBH harbor a mutation of the DCX gene, leaving the genetic causes unexplained, and genetic counseling problematic, in the remaining patients. Our purpose was to verify the extent to which exonic deletions or duplications of the DCX gene would account for sporadic SBH with A>P gradient but normal gene sequencing., Methods: We identified 23 patients (22 women, 1 man) with sporadic, diffuse, or anteriorly predominant SBH. After sequencing the DCX gene and finding mutations in 12 (11 women, 1 man), we used multiplex ligation-dependent probe amplification (MLPA) to search for whole-exon deletions or duplications in the 11 remaining women. We used semiquantitative fluorescent multiplex PCR (SQF-PCR) and Southern blot to confirm MLPA findings., Results: MLPA assay uncovered two deletions encompassing exons 3 to 5, and one involving exon 6, in 3 of 11 women (27%) and raised the percentage of DCX mutations from 52% to 65% in our series. SQF-PCR performed in all three women and Southern blot analysis performed in two confirmed the deletions., Conclusions: MLPA uncovers large genomic deletions of the DCX gene in a subset of patients with SBH in whom no mutations are found after gene sequencing. Deletions of DCX are an underascertained cause of SBH.

Rationale: There is increasing interest in the potential influence of fetal and early life conditions on childhood wheezing., Objectives: To investigate the associations between maternal complications and procedures in pregnancy and at birth and the risk of various wheezing phenotypes in young children., Methods: We studied 15,609 children, aged 6-7 yr, enrolled in a population-based study. Standardized questionnaires were completed by the children's mothers., Results: Of the children, 9.5% (1,478) had transient early wheezing, 5.4% (884) had persistent wheezing, and 6.1% (948) had late-onset wheezing. Maternal hypertension or preeclampsia was associated with an increased risk of all three wheezing phenotypes (for transient early wheezing: odds ratio [OR], 1.40; 95% confidence interval [95% CI], 1.08-1.82; for persistent wheezing: OR, 1.59; 95% CI, 1.15-2.19; and for late-onset wheezing: OR, 1.47; 95% CI, 1.06-2.01). Use of antibiotics for urinary tract infections was associated with transient early wheezing (OR, 1.52; 95% CI, 1.16-2.00), whereas antibiotic administration at delivery was associated with both transient early wheezing (OR, 1.21; 95% CI, 1.01-1.46) and persistent wheezing (OR, 1.39; 95% CI, 1.10-1.75). Children who had a mother with diabetes were also more likely to have persistent wheezing (OR, 1.72; 95% CI, 0.99-3.00). Neither amniocentesis/chorionic villus sampling, nor weight gain in pregnancy, nor cesarean section was associated with the subsequent development of wheezing. Maternal asthma or atopy was not an effect modifier of the associations found., Conclusions: Some maternal complications during pregnancy and at delivery may increase the risk of developing different phenotypes of wheezing in childhood.

Purines and pyrimidines are the basic constituents of DNA and RNA and constitute the basis of at least 50 other important compounds that serve equally vital but separate roles as integral components of intracellular mononucleotide pools. They maintain the supply of these basic components to the different nucleotide pools through an extremely efficient mechanism involving the degradation and recycling of the daily waste products of normal cell turnover. We have developed an LC-MS/MS diagnostic and routine monitoring method for known defects due to both purine and pyrimidine metabolism in a single analysis. Precision tests were made by spiking several urine samples with different creatinine concentrations. For nonspiked low-creatinine urine, intraday precision was in the range of 0.1-9.8% and interday precision was between 1.6 and 14.1%. For nonspiked high-creatinine urine, intraday precision was in the range 0.5-17.2% and interday precision was between 1.5 and 29%. Limit-of-detection (LOD) was in the range 0.1-10 micromol/l and limit-of-quantification (LOQ) in the range of 0.2-15 micromol/l. The current 'dilute and shoot' approach monitors many metabolites, and utilizes a reverse phase chromatographic analysis with a detection requiring 17 min of analysis time. Tandem mass spectrometry and isotope dilution technique enable the accurate quantitation of more than 30 metabolites in one analysis., (Copyright 2006 John Wiley & Sons, Ltd.)

Reported here is the case of a 6-week-old female infant with a severe Bordetella pertussis infection requiring supportive pressure-positive ventilation in the intensive care unit. After being discharged from the intensive care unit, she developed hemolytic anemia, thrombocytopenia and acute renal failure, which suggested a diagnosis of hemolytic uremic syndrome. The clinical outcome was favorable with no renal consequences. This case suggests there may be a direct cause-effect relationship between B. pertussis infection and hemolytic uremic syndrome.

Sirenomelia is an extremely complex and rare malformation with different degrees of lower-extremities fusion associated with gastrointestinal, musculoskeletal, vascular, cardiopulmonary, and central nervous system malformations. In the English literature, there are only 5 reports of infants surviving with this condition. In our case, a 2540-g female infant was born with normal vital signs, no facial dysmorphism, and a complete soft tissue fusion of the lower limbs, from perineum to ankles. Radiologic examinations revealed an intestinal atresia and a single pelvic kidney, with a unique ureter, 2 femurs, 2 tibias, 2 fibulas, and 2 feet (simpus dipus). At 7 months of age, a multidisciplinary surgical team achieved complete separation of the lower limbs, with independent vascular and nerve supplies. At the time of this writing, the infant was 28 months old and had a regular growth curve. Many future reconstructive surgeries have been planned to achieve an acceptable quality of life for this infant.

In this report we present the case of a 15-month-old girl with hyponatraemic-hypertensive syndrome (HHS) caused by stenosis of the left renal artery. On sonographic examination the contralateral non-stenotic kidney appeared enlarged and with cortical hyperechogenicity mimicking a parenchymal lesion. After successful percutaneous transluminal angioplasty, when the girl became normotensive, her serum electrolyte and acid-base balance became normal within a few days. The contralateral non-stenotic kidney hyperechogenicity also disappeared, but only after a period of 6 months, suggesting parenchymal damage due to tubulointerstitial injury, even though reversible. Our case confirms that renovascular hypertension may rarely also be present with HHS in children and that metabolic and morphological alterations are reversible after the resolution of arterial stenosis.

The patients affected by vitamin B12-unresponsive methylmalonic acidemia (MMA) on the long run develop chronic renal disease with interstitial nephropathy and progressive renal insufficiency. The mechanism of nephrotoxicity in vitamin B12-unresponsive MMA is not yet known. Chronic hyporeninemic hypoaldosteronism has been found in many cases of methylmalonic acidemia, hyperkalemia and renal tubular acidosis type 4. We report 2 patients affected by B12-unresponsive methylmalonic acidemia diagnosed at the age of 23 months and 5 years, respectively, with normal glomerular filtration and function. They showed hyporeninemic hypoaldosteronism and significant hyperkalemia requiring sodium potassium exchange resin (Kayexalate) therapy after an episode of metabolic decompensation leading to diagnosis of MMA. In both children, hyporeninemic hypoaldosteronism and hyperkalemia disappeared after 6 months of good metabolic control.

We examined the possibility of using microsatellite and mitochondrial DNA polymorphisms as markers to detect the clonal origin of tumor cells found in the same patient. We considered two children with complex tumor diseases: one with supratentorial primitive neuroectodermal tumors (PNET) and a hepatic rhabdoid tumor and another with brain and abdominal rhabdoid tumors. In the first patient we found an mtDNA cytosine insertion both in the normal tissue and in the primary tumor, whereas in the hepatic tumor we detected an insertion of 2 cytosine. In the second child, who had a constitutional mutation of hSNF5/INI-1, we identified the same mtDNA pattern both in normal tissue and in the abdominal tumor but not in the brain tumor, which presented three different mtDNA polymorphisms. Thus, we demonstrated the same clonal origin for tumors in the first patient and different clonal origins of the tumors in the second patient. At times it is very difficult to discriminate two neoplastic lesions or metastatic diseases by using only histopathologic techniques. Molecular examination of clonality is a useful tool to obtain information about the origin of synchronous and/or metachronous tumors found in the same patient.

Background: Growth hormone (GH) treatment in patients with GH deficiency (GHD) can determine changes in the thyroid function. The clinical significance of these changes remains controversial, and all studies have so far covered rather a short period--usually no longer than one year., Objective: To determine the effect of long-term recombinant hGH treatment in children with idiopathic GHD on the thyroid function., Patients and Methods: Nineteen prepubertal children (12 boys and 7 girls, mean age 9.2 +/- 3.1 years) with idiopathic GHD were studied and followed for twenty-four months. None of the patients showed multiple pituitary hormone deficiencies. Nineteen healthy children matched for age and sex acted as controls., Results: Patients with GHD showed a significant increase in TT (3) at twelve months and in FT (3) at six and twelve months after starting GH treatment, with a significant decrease at eighteen and twenty-four months. TT (4) level decreased significantly at twelve months and increased significantly at eighteen and twenty-four months. FT (4) also decreased, but only slightly, after twelve months of hGH treatment, and then increased significantly at twenty-four months. TSH levels did not vary significantly during the course of therapy. TT (3)/TT (4) and FT (3)/FT (4) ratios increased significantly after six and twelve months of therapy and significantly decreased later, approaching pre-therapy values. The SDS of Growth Velocity (SDS-GV) increased remarkably during the first year of therapy and then decreased significantly during the second year, although it remained significantly higher than the pre-therapy values. TT (3) and TT (3)/TT (4) ratio displayed a significant correlation with SDS-GV at twelve months of therapy. In a multiple regression analysis with age, bone age, parental height, GH dose, TT (3,) TT (3)/TT (4), and the SDS of IGF-I, only the TT (3)/TT (4) ratio at twelve months of therapy (p < 0.001) was identified as a significant predictor of SDS-GV., Conclusion: Our data confirm that changes in thyroid function are present in GHD children during long-term hGH therapy. These changes probably resulted from the effect of hGH on the peripheral metabolism of thyroid hormones and appear to be transitory, disappearing during the second year of hGH treatment. We speculate on the functional significance of these changes, and in particular, on their role in catch-up growth after hGH therapy.

Objective: Children and adolescents with type 1 (insulin-dependent) diabetes mellitus (T1DM) show several impairment of bone metabolism and structure, resulting in a higher risk of decreased bone mass and its related complications later in life. Alterations of the nuclear factor-kappaB ligand (RANKL)/osteoprotegerin (OPG) system have been implicated in several metabolic bone diseases characterized by increased osteoclast differentiation and activation and enhanced bone resorption., Design: We aimed to assess OPG levels and to investigate the possible relation between OPG levels, bone status and glycemic control in a group of prepubertal children with T1DM without microvascular complications., Methods: Twenty-six prepubertal T1DM children (median age 9.9 years, range 4.1-13.1 years) were studied. In all patients, serum OPG, hemoglobin (Hb)A1c, parathyroid hormone (PTH) and 25-dihy-droxyvitamin D (25-D) levels were evaluated. Bone quality was determined by measuring the attenuation of ultrasound waves by bone (broadband ultrasound attenuation (BUA)) at the calcaneal site. The data were compared with those of a group of 45 age-, sex-and body-size-matched healthy children., Results: Children with T1DM showed a reduced Z-score BUA in comparison with the control group (Student's t-test, P < 0.0001). Plasma OPG levels were significantly higher in diabetic children than in controls (Student's t-test, P < 0.0001). In T1DM children, Z-score BUA values displayed a significant correlation with OPG (Student's t-test, r = -0.62; P = 0.001), and HbA1c (r = -0.59; P = 0.007). OPG levels were significantly correlated with HbA1c (r = 0.56; P = 0.008). In a multiple regression analysis including age, duration of diabetes, physical activity, calcium intake, mean HbA1c and Z-score BUA, only HbA1c significantly predicted serum OPG levels (beta 0.67; P = 0.003)., Conclusions: Prepubertal children with T1DM have a significant increase of OPG levels. OPG serum concentrations are correlated to calcaneal BUA and HbA1c values. OPG could be a new marker of reduced bone mass in children with T1DM.

A 5-month-old girl with distal renal tubular acidosis (RTA) and hyperammonaemia that had lasted for 12 days, despite metabolic acidosis correction, is presented in this report. The patient showed failure to thrive, poor feeding, hypotonia and vomiting crisis in absence of inborn errors of metabolism. Probably, hyperammonaemia was the result of an imbalance between the increased ammonia synthesis, in response to metabolic acidosis, and the impaired ammonia excretion, typical of distal RTA. Our case confirms that hyperammonaemia may be observed in distal RTA, mimicking an inborn error of metabolism, and it underlines that hyperammonaemia may persist several days after metabolic acidosis correction.

The prevalence and the clinical features of chronic daily headache (CDH) were studied in 968 children and adolescents observed during a period of one year in the Headache Centre of the Anna Meyer Paediatric Hospital of Florence. Nine hundred and forty-four patients (97.52%) had primary headache according to ICHD-II, 24 subjects had secondary headache and 56 patients had CDH (5.93% of primary headaches). The mean age of subjects with CDH was higher than general (13.5 vs. 11.5 years), with a female preponderance (69.6% vs. 30.4%). According to the ICHD-II, headaches were classified as chronic migraine in 10 patients (1.5.2 ICHD-II), chronic tension-type headache in 36 (2.3 ICHD-II), new daily persistent headache in 8 (4.8 ICHD-II) and 2 patients reported mixed pattern (chronic migraine+chronic tension type headache). Medication overuse was not implicated in our patients.

Non-expensive and low-complexity surrogate markers for monitoring the response to combined antiretroviral therapy (combined-ART) are needed in poor-resource settings where routine assessment of CD4+ T-lymphocyte count and viral load can not be afforded. We longitudinally evaluated Ig serum levels in 234 HIV-1 infected children receiving combined-ART with > or = 3 drugs. Since Ig levels physiologically vary with age, differences at different age periods were evaluated as differences in z-scores calculated using the mean and standard deviation of the normal population for each age period. Data from 17 (7.3%) children with immunological failure and from 54 (23.1%) children with virological failure of combined-ART were compared with data from not-failed children. At baseline children with immunological failure showed higher IgM z-scores (P = 0.042) than children without. After 3-12 months of therapy immunologically failed children displayed higher viral loads (P < 0.0001) and IgA (P = 0.043) z-scores than not-failed children. Similarly, at the same follow-up time, children with virological failure showed lower CD4(+) T-lymphocyte percentages (P = 0.005) and higher IgA z-scores (P < 0.0001) than not-failed children. No difference in IgG or IgM z-scores was evidenced between failed and not-failed children after 3-12 months of therapy. In conclusion, IgA serum level is a cheap and low-complexity marker of immunological or virological failure of combined-ART which might be adopted in poor-resource settings.

Live attenuated vaccines are usually contraindicated in patients with congenital or acquired immunodeficiency. On the other hand, infections due to wild type virus may be particularly severe in patients with low levels of T cells. The aim of the present study was to evaluate safety and immunogenicity of measles-mumps-rubella (MMR) vaccine in children with congenital T cell defect (DiGeorge anomaly). Fourteen patients were included in the study. No severe adverse reaction was reported. No difference between patients and controls was found in frequency of seroconversion for both measles (92.9% versus 96.3%) and rubella (92.9% versus 100%). No difference in mean titres of anti-measles (1.62+/-0.54 versus 1.89+/-0.49 index) (p=0.13) or anti-rubella (78.1+/-48.0 versus 72.0+/-41.0 UI/ml, p=0.68) antibodies was found between patients and controls. No decrease in CD4 cells was detected after immunization. MMR vaccine is immunogenic and can be safely used in patients with DiGeorge anomaly, so preventing severe complication due to wild virus infection.

Chronic pulmonary infections caused by Pseudomonas aeruginosa, Burkholderia cepacia complex and Staphylococcus aureus are responsible for most of the morbidity and mortality of patients with cystic fibrosis (CF). Little is known about the routes of transmission of these pathogens from environmental or hospital sources to the patients. We hypothesised that strains of P. aeruginosa, B. cepacia complex and methicillin-resistant S. aureus (MRSA) are nosocomially acquired by CF patients. Bacterial isolates were obtained from 164 patients attending the CF Centre of Florence and from the hospital environment and the strains typed using restriction enzymes and pulsed-field gel electrophoresis (PFGE). Seventy (43%) of patients were colonised by P. aeruginosa, 6 (3.6%) by B. cepacia complex, and 11 (7%) by MRSA. Three P. aeruginosa strains were isolated from the sinks of the ward. All the MRSA isolates differed from each other. The analysis of 83 P. aeruginosa strains showed identical genotypes in five pairs of patients, whereas from the six patients infected with B. cepacia complex strains, two patients harboured identical genotypes. These pairs of patients had no contact with each other outside the CF centre and P. aeruginosa genotypes from the hospital environment differed from these clinical isolates, suggesting a possible common source of infection within or outside the centre. The study showed that, despite isolation precautions, a minimal risk of cross-infection still existed in the CF centre and that hygienic standards should be increased to further reduce this risk.

Objective: To provide centiles for birth weight (BW) according to gestational age (GA) and sex for infants born in Italy., Methods: We used records of the whole neonatal population of Tuscany, a region in Italy, from July 1991 to June 2002 as resulting from the database of the cystic fibrosis neonatal screening program (n=290129). We excluded as unlikely for GA those BW that were more than two interquartile ranges above the 75th centile or below the 25th centile for each GA and gender group., Results: We present the 3rd, 10th, 25th, 50th, 75th, 90th and 97th centiles of BW for GA from the 24th to 43rd week of gestation for male and female Italian neonates, as both tables and smoothed curves., Conclusions: The large size of the examined population allows us to provide up-to-date, reliable BW for GA centiles for Italian newborns, especially for lower GAs.

We describe the first known case of congenital lymphoedema associated with selective deficit of naïve CD4+ T-lymphocytes. A high proportion of naïve CD4+ T-lymphocytes was found in the ascitic fluid, supporting the hypothesis of extra-vascular sequestration of these cells into lymphoedematous tissue.

We report the modulating action of the L436F new polymorphism identified in the GLB1 gene of a patient affected by GM1 gangliosidosis with onset at 17 months and rapidly progressive psychomotor deterioration. Sequencing analysis and familial restriction studies revealed that the maternal allele of this patient carried the L436F polymorphism in cis with the known R201C mutation. The new mutation R68W was identified in his paternal allele. Since the GLB1 activity of the patient's leukocytes was very low and compatible with both the type-I and the type-II form of the disease, the potential impact of each mutation was investigated by expression studies in COS1 cells, and Western blots. Expression study of the R68W mutated allele resulted in no GLB1 activity. Transfection with a vector carrying the R201C mutation gave rise to a residual GLB1 activity, which, interestingly, was severely reduced in transfection with the L436F/R201C allele. These expression studies, together with co-transfection experiments, suggest that the R201C/L436F GLB1 "complex allele" leads to this patient's clinical and biochemical findings. The type-II phenotype of the disease is subdivided into late infantile and juvenile forms. The clinical and molecular characterization of this patient as late-infantile GM1 gangliosidosis is in keeping with a clear-cut division between the two sub forms of the type-II phenotype. The modulating role of the L436F polymorphism should be stressed as a cause of this patient's condition. This model suggests that the combination of missense mutations or polymorphisms should be evaluated when diagnosing inherited genetic disorders.

We have recently introduced a new protocol for analyzing all core loci of the Federal Bureau of Investigation's (FBI) Combined DNA Index System (CODIS) with an infrared (IR) automatic DNA sequencer (LI-COR 4200). The amplicons were labeled with forward oligonucleotide primers, covalently linked to a new infrared fluorescent molecule (IRDye 800). The alleles were displayed as familiar autoradiogram-like images with real-time detection. This protocol was employed for paternity testing, population studies, and identification of degraded forensic samples. We extensively analyzed some simulated forensic samples and mixed stains (blood, semen, saliva, bones, and fixed archival embedded tissues), comparing the results with donor samples. Sensitivity studies were also performed for the four multiplex systems. Our results show the efficiency, reliability, and accuracy of the IR system for the analysis of forensic samples. We also compared the efficiency of the multiplex protocol with ultraviolet (UV) technology. Paternity tests, undegraded DNA samples, and real forensic samples were analyzed with this approach based on IR technology and with UV-based automatic sequencers in combination with commercially-available kits. The comparability of the results with the widespread UV methods suggests that it is possible to exchange data between laboratories using the same core group of markers but different primer sets and detection methods.

Background: A predominance of Th2 response has been suggested in vernal keratoconjunctivitis (VKC), and a high prevalence of IgE-sensitized (IgE-S) patients has been reported (positive skin prick test or serum-specific-IgE). Palpebral and bulbar VKC are considered to be expressions of the same disease and only occasional racial and histopathological differences are described between the two forms. Tear levels of eosinophil cationic proteins have been correlated with the severity of ocular symptoms; however, there is no published study that demonstrates the presence of serum markers of disease activity., Objective: This study was performed to evaluate the prevalence of IgE-sensitization in palpebral, bulbar and mixed VKC and to determine possible useful markers of disease activity in peripheral circulation., Methods: A total of 110 white VKC patients (mean age 8.3 years, range 3.2-18 years) were evaluated for ocular score in the active phase of the disease. Skin prick tests and serum-specific IgE for common allergens, serum-total IgE, peripheral blood eosinophil counts (PBECs) and serum eosinophil cationic protein (s-ECP) were determined. Fifteen age-matched non-IgE-S control children underwent the same determinations., Results: s-ECP, PBECs and s-total IgE were significantly higher in IgE-S than in non-IgE-S VKC patients and in non-IgE-S VKC patients than in controls. A lower prevalence of IgE-S patients was found in bulbar vs. tarsal (P = 0. 050) or mixed forms (P = 0.002). The score of giant papillae was strongly correlated with s-ECP levels (P < 0.001) and with PBECs (P = 0.001)., Conclusions: Our data suggest that an overall eosinophilic response is present in VKC independently of IgE-sensitization; bulbar forms, unlike tarsal and mixed forms, were associated with a low prevalence of IgE-sensitization. Serum ECP was a useful marker of disease activity in tarsal and mixed forms.