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Pediatric cholelithiasis is being increasingly diagnosed owing to the widespread use of ultrasonography, raised pediatric obesity and use of long-time parenteral nutrition. Clinical presentation is variable and complicated onset could lead to severe consequences. The aim of this study is to present a 15-years-experience of a pediatric third-level-center in treating cholelithiasis. A retrospective study collecting children with gallstone operated on between 2006 and 2020 is presented. Demographic data, clinical presentation, sonographic findings, risk factors, surgery, complications, follow-up were evaluated. 199 patients were included. Twenty-nine patients (14.5%) with cholelithiasis had a complicated onset. Patients with cholelithiasis older than 10 years had a higher rate of symptomatic/complicated onset. Hemolytic disease was the most frequent co-morbidity (16.1%). Laparoscopic cholecystectomy was performed in 192 cases (96.5%) with a conversion rate of 1.6%. Nearly 1% showed a major post-operative complication after cholecystectomy. Cholelithiasis in patients older than 10 years can be safely treated with laparoscopic cholecystectomy, especially if one or more co-morbidities are present. Laparoscopic splenectomy can be easily associated in the same procedure depending on medical indication. Definition of pediatric surgical timing guidelines would be of quite interest in the field.

ABSTRACTGenus Menthais represented in Morocco by 10 species including hybrids. The species of this genus are used as herbal teas and condiments, due to their distinct aroma. In this study, infusions of the fresh aerial parts of three Menthaspp., commonly cultivated and used in Morocco, were evaluated for phenolic contents and antioxidant activity. Antioxidant capacity was assessed by three tests: scavenger activity by DPPH, FRAP (Ferric Reducing Antioxidant Power), and β-carotene bleaching methods. Infusions of M. piperitapossess the best ability to reduce DPPH and chelate iron (IC50 = 7.50 ± 0.19 and 5.84 ± 0.36 μg/mL, respectively), while M. spicatashows great efficiency on inhibiting β-carotene (IC50 = 62.67 ± 0.01 μg/mL). The antioxidant capacity was significantly correlated with the total phenolic content and HPLC chromatographic analyses showed the predominance of phenolic acids, especially rosmarinic acid. Furthermore, by GC-MS analysis of Menthaspp. essential oils, eucalyptol, limonene, carvone, and pulegone were identified as predominant components.

Purpose: Hirschsprung’s disease (HSCR), anorectal malformations (ARM), and inflammatory bowel diseases (IBD) are the main paediatric colorectal diseases treated with minimally invasive surgery (MIS). The authors describe their experience and track the historical evolution of MIS in these entities. A non-systematic review is performed to determine the incidence and type of complications reported  to date. Methods: The literature research was performed using PubMed database. The personal series included all patients with HSCR, ARM, or IBD who underwent MIS from 2006 to 2018. Demographic data, type of surgical procedure, complications and long-term outcomes were analysed. Results: The literature research showed an increasing use of MIS for HSCR over the years, with less post-operative pain, shorter hospital stay, and improved  cosmesis compared to open surgery; without differences in complication rates and outcomes. Only high ARMs are considered for managment by MIS; with laparoscopy, however being a risk factor  for the development of  a remnant of the original fistula (ROOF) and rectal mucosal prolapse. MIS procedures have shown excellent results in adults affected by IBD, whereas only a few studies have been published on paediatric patients. We have performed 269 laparoscopic colorectal procedures; half of them for HSCR with 3 major complications (2.3%). Twenty-one laparoscopic procedures for ARM were associated with 1 anastomotic stricture and 1 ROOF. One hundred and fourteen laparoscopic procedures were performed for IBD, with 13.1% complication. Conclusions: Minimally invasive surgery provides similar results when compared to total trans-anal approach and open surgery in HSCR. Similarly, its use in ARM and IBD seems to be safe and beneficial, provided that selected cases are performed by highly experienced surgeons.

The objective of the present study is to analyze prognostic factors affecting survival of patients receiving stereotactic radiosurgery (SRS) for second brain metastatic event (SBME) following initial treatment with whole brain irradiation (WBI), surgical resection, or previous SRS. The 88 patients treated with SRS for SBME at Philadelphia CyberKnife between January 2006 and October 2013 were included in the study group. Cox proportional-hazards regression was used to identify prognostic factors that significantly impacted survival from the time of SRS for SBME. Independent variables considered in survival analysis included primary disease, first brain metastatic event (FBME) treatment type, age, gender, number of brain metastases at SBME, Karnofsky performance status (KPS), recursive partitioning analysis (RPA), and presence of extracranial metastasis. The median survival for all patients was 7.31 months. Log-rank comparison of Kaplan-Meier survival curves revealed significant impact by Karnofsky performance status (p= 0.003), RPA class (p= 0.008), age (p= 0.014), and FBME treatment type (p= 0.010). Median survival was longer for patients who had not previously received WBI (14.7 months). Median survival was further increased in patients who had not received previous WBI and demonstrated KPS scores of 70–100 (19.5 months). Patients who received WBI prior to SBME treatment experienced a pronounced decrement in median survival (5.7 months), yet patients in this group who demonstrated strong KPS scores (80–100) experienced significantly increased survival (15.5 months). The outcomes of SRS for SBME are most favorable for patients who have not received previous WBI or who have maintained higher performance status despite previous WBI.

Mercaptopurine (MP) and azathioprine (AZA) are thiopurine drugs that are used in paediatrics for the treatment of inflammatory bowel disease (IBD), Therapeutic drug monitoring (TDM) of thiopurine active metabolites, 6-methylmercaptopurine (6MMP) and 6-thioguanine nucleotides (6-TGN) is a common practice for the assessment of toxicity surveillance and treatment optimization. In this paper we described the development and validation of a new High Performance Liquid Chromatography coupled to Diode Array Detection (HPLC-DAD) method for the simultaneous quantification of 6-TGN and 6-MMP in whole blood with a total runtime of 7.5 minutes over a wide range of concentrations (50 – 2000 pmol/8x10^8 RBC for 6-TG and 20 – 15000 pmol/8x10^8 RBC for 6-MMP respectively). The new method has been validated following international guidelines on bioanalytical method validation. Within- and between-run precision and accuracy results for quality control samples were always ≤ 15% of the nominal concentrations. The correlation with our reference method was excellent. The new HPLCDAD method that we describe is robust, rapid, cost effective and suitable for application to the routine TDM analyses.

Therapeutic drug monitoring (TDM) of major metabolites of thiopurine drugs is a widely used tool for assessing treatment efficacy and toxicity in patients with inflammatory bowel disease (IBD). We report the laboratory and clinical validation of a simple and reliable high performance liquid chromatography (HPLC) method for the measurement of 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP) on paediatric patients with IBD. The aim of this paper is to develop and validate a method for the measurement of 6-TGN and 6-MMP applicable to routine practice and to evaluate the usefulness of the TDM of thiopurine drugs in children with IBD attending our Gastroenterology Unit. The HPLC method was validated following international guidelines starting from red blood cells (RBC) and whole blood (WB). A comparison between RBC and WB was assessed. The usefulness of TDM was then evaluated using the new method from WB in 47 paediatric patients with IBD treated with thiopurine drugs. WB and RBC resulted in interchangeable matrices. The majority of patients had the metabolite levels inside the therapeutic ranges. A moderate correlation was found between 6-MMP concentration and the dose of thiopurines. A higher percentage of non responders was found among patients with lower levels of 6-TGN. Toxicity was found in eight patients and was evaluated in respect to the metabolite concentration. The described HPLC method is applicable to routine practice and it is suitable for its use in multicentric studies. Our results of TDM on paediatric IBD patients can contribute to clarify its role in their therapeutic management.

The aim of this study was to correlate the data obtained with multiple intraluminal esophageal impedance and pH (MII-pH) recordings in infants and children referred for suspected gastroesophageal reflux disease with esophageal histology. In a prospective study, results of esophageal biopsies and MII-pH recording obtained in 45 children (mean age ± SD: 69 ± 55 months) were analyzed. Regarding the MII-pH data, an automatic (Autoscan Bioview Analysis Software, version 5.3.4, Sandhill Scientific Inc, Highlands Ranch, CO) and a manual reading were performed; an automatic pH analysis (meal included) was also performed. Acidic, weakly acidic, and alkaline reflux episodes accounted, respectively, for 48.7%, 49.5%, and 1.8% of the total number of reflux episodes detected by MII-pH. Esophagitis was present in 25 (56%) children. Concordance between classic pH-study analysis (alone) and esophageal histology was found in 19 of 45 (42%) children. According to the MII-pH analysis, the mean and median value of the pH were significantly higher in the group with esophagitis than in the group with normal esophageal histology. A longer clearance time was found in the group with esophagitis than in subjects with normal histology. Gas reflux episodes represented 21% of the total reflux episodes and were comparable in both groups. Multiple intraluminal esophageal impedance and pH analysis does not provide a distinct parameter to predict esophageal mucosal injury in children. In our population, MII-pH shows comparable acidic, weakly acidic, alkaline, and gas reflux in children with and without esophagitis. Further research is needed to analyze clearance parameters.

The aim of this study was to correlate the data obtained with multiple intraluminal esophageal impedance and pH (MII-pH) recordings in infants and children referred for suspected gastroesophageal reflux disease with esophageal histology.

AbstractBackground: Headache is a disease that has a high social impact in the paediatric as well as in the adult population, often resulting in a significant reduction in the young patient’s quality of life, reflected primarily in a greater number of days off school and increasingly frequent recourse to symptomatic drugs. The idea for this study came from the clinical impression that some paediatric headache patients might benefit more from inpatient than outpatient care. Aim: The aim of our study was to compare the effectiveness of hospitalization to outpatient care of patients with newly diagnosed frequent and disabling headache. Methods: A pragmatic randomized open-label trial was conducted at the Child Neurology Clinic of the University of Pavia, Italy. Children and adolescents with a 2- to 6-month moderate-to-severe migraine or tension-type headache history were randomized to hospital admission or outpatient assessment and followed for 6 months. The efficacy of the two therapeutic strategies was measured by counting the number of responders in each arm. Other end points included the mean frequency and duration of attacks, the number of drug prescriptions taken to control pain, and the number of patients and physicians expressing satisfaction with treatment. Results: The study population included 27 girls and 23 boys aged 8 through 18 years with migraine (23 cases) or tension-type headache (27 cases). Compared to outpatient assessment, hospital admission was correlated to a significant increase in the number of responders: 0 vs. 44% (1 month), 0 vs. 68% (3 months), and 12 vs. 68% (6 months). The mean frequency and duration of attacks were significantly lower in hospitalized patients (p < 0.0001). Hospitalization was correlated with a significant reduction of patients with severe headache (p < 0.005), a reduction of drug use, and a higher number of satisfied patients and physicians (p < 0.05). Logistic regression analysis confirmed the higher responder rate among hospitalized patients after adjusting for age, sex, diagnosis, and headache characteristics or admission. Conclusions: We think hospitalization reduces the emotional mechanisms that provoke stress in children and often induce or favour headache attacks. If these mechanisms can be interrupted, the management of disease may become easier and with enduring benefits.Copyright © 2007 S. Karger AG, Basel

Described are the outcomes of 11 Italian patients with Aicardi–Goutières syndrome. Neurologic symptoms progressed in the first year of life and stabilized by the end of the second year in 10 patients. White matter abnormalities remained stable; cerebral atrophy was stable in four patients and progressive in two. Calcifications increased (in number and size) in two of six patients. Serial CSF and serum interferon- measurements (three patients) showed reduced CSF interferon- levels.

We describe the clinical and neuroradiological features of eleven patients with Aicardi-Goutières syndrome, a rare and severe progressive encephalopathy with onset in the first year of life. The syndrome is autosomal recessive with varying clinical presentation and course. Our patients were studied by CT and MR imaging and findings were in agreement with literature reports. Calcification of the basal nuclei was found in 100% of cases and six patients presented a progressive increase in the number and size of the calcifications which were bilateral and largely symmetrical. White matter changes were seen in 76% of cases without a specific pattern of distribution. Early neuroradiological diagnosis of suspect Aicardi-Goutières syndrome is established by ruling out other pathological processes and the site and features of the calcifications rather than the white matter changes is important to then search for typical CSF changes.

Human immunodeficiency virus type 1 (HIV-1) expresses the Vif, Vpr, Vpu, and Env proteins through complex differential splicing of a single full-length RNA precursor. We used HIV-1-specific oligonucleotide primer pairs in a quantitative polymerase chain reaction procedure on RNA from fresh peripheral blood lymphocytes infected with HIV-1JR-CSF to detect and characterize the singly spliced RNA species which might encode these proteins. The nucleotide sequences at the junctions of splice donor and acceptor sites of these RNAs were determined. One of these RNAs, which has not been previously described, appears to be a novel HIV-1 RNA encoding Env and/or Vpu proteins.

In the absence of Rev or the Rev-responsive element, the Rev-dependent human immunodeficiency virus type 1 (HIV-1) RNAs do not behave as mRNAs; rather, they exhibit nuclear defects in splicing and/or nuclear export and cytoplasmic defects in stability and translation. A translational initiation factor, eIF-5A, has recently been shown to bind specifically to the Rev activation domain. As the binding of poly(A)-binding protein 1 (PAB1) to the poly(A) tail of mRNAs is involved in both the stability and translation of cytoplasmic mRNAs, we investigated whether Rev might influence the association of PAB1 with cytoplasmic HIV-1 RNAs. Antibodies were generated against PAB1. We used these antibodies in an immunoprecipitation assay to detect specific binding of PAB1 to cytoplasmic mRNAs. We found that in the presence of Rev, PAB1 was associated with Rev-dependent and Rev-independent RNAs in the cytoplasm of transfected cells. However, in the absence of functional Rev, we found little or no PAB1 associated with Rev-dependent RNAs. These RNAs were capable of binding PAB1 in vitro. These results demonstrate that HIV-1 RNAs are defective in PAB1 association in the absence of Rev.

Constructs were generated in which expression of human immunodeficiency virus type 1 (HIV-1) protease (PR) was placed under control of the HIV-1 long terminal repeat, thus requiring the HIV-1 Tat protein for expression of PR. The activity of PR was assessed by cotransfection with a construct producing a Gag substrate. Expression of PR as an intramolecular multimer resulted in a large increase in PR activity in comparison with the level obtained with the expression of PR as a monomer. A cytotoxic effect of PR expression was also exhibited by the constructs expressing PR multimers. CD4+ T-cell lines were generated with a construct producing PR as a linked tetramer and screened for PR activity and inducibility. The replication of HIV-1 in these cell lines was several orders of magnitude reduced in comparison with that in cell lines not expressing PR. Infection in these cell lines could be detected early after infection but disappeared over time. Infection of the PR-expressing cell lines could be increased several orders of magnitude by the addition of a specific inhibitor of PR, U75875 (Upjohn), after infection of the cells, demonstrating that the potent inhibition of HIV-1 replication in these cells was directly due to the expression of PR.

Different strains of human immunodeficiency virus type 1 (HIV-1) vary in the ability to replicate in cells that bear the HIV-1 receptor, CD4. The mechanism responsible for these cell tropism differences is unknown. We examined different isolates of HIV-1 with regard to replication in specific tumor-derived CD4-positive T-cell lines and normal peripheral blood lymphocytes. To investigate early events in the virus life cycle at low multiplicities of infection, we used a modification of the polymerase chain reaction method. Use of a molecularly cloned primary HIV-1 isolate, HIV-1 JR-CSF, restricted for replication in T-cell lines, demonstrated that little or no viral DNA or RNA was synthesized in nonpermissive cells after infection. However, transfection of proviral DNA resulted in efficient transient virus production from these cells. Therefore, we conclude that at least one block to infection for HIV-1 strains in nonpermissive T cells occurs at a point in entry or uncoating before provirus formation.

A cis-acting enhancer element has been detected within the gag gene of several avian retroviruses, including Rous sarcoma virus, Fujinami sarcoma virus, and the endogenous Rous-associated virus-0. A consensus enhancer core sequence, GTGGTTTG, is present in all of these viral genomes, approximately 900 bases downstream from the site of initiation of transcription. When an internal fragment derived from the gag gene of any of these viruses (spanning nucleotides 533 to approximately 1149) was inserted into a plasmid containing the chloramphenicol acetyltransferase (cat) gene under control of the simian virus 40 promoter, 9- or 21-fold enhancement of CAT expression was observed after transfection into mouse L cells and chicken embryo fibroblasts, respectively. This enhancement was not dependent on the position of insertion of the gag fragment into the plasmid. However, there was a strong dependence on orientation, with higher levels of CAT expression in constructs in which the 5' end of the gag fragment was nearest to the promoter, suggesting a possible negative regulatory element at the 3' end of this fragment. Deletion of the 3' end of the insert resulted in a gag fragment, containing nucleotides 533 to 1017, which enhanced expression equally in either orientation. When the gag fragment was inserted into a plasmid containing the cat gene under the control of an intact Rous sarcoma virus long terminal repeat, it induced a two- to threefold increase in CAT activity and CAT mRNA levels. Translation of the gag fragment did not appear to be necessary for the observed enhancement, since two insertional mutations resulting in frameshifts in the gag insert did not affect CAT expression. However, deletion of a 330-base internal fragment from the gag insert restored a basal level of CAT activity. These results suggest that retroviruses have regulatory elements within their genes distinct from those in the long terminal repeats that flank the genes.

The human T-cell leukemia viruses (HTLVs) encode a trans-regulatory protein, Rex, which differentially regulates viral gene expression by controlling the cytoplasmic accumulation of viral mRNAs. Because of insufficient amounts of purified protein, biochemical characterization of Rex activity has not previously been performed. Here, utilizing the baculovirus expression system, we purified HTLV type II (HTLV-II) Rex from the cytoplasmic fraction of recombinant baculovirus-infected insect cells by heparin-agarose chromatography. We directly demonstrated that Rex specifically bound HTLV-II 5' long terminal repeat RNA in both gel mobility shift and immunobinding assays. Sequences sufficient for Rex binding were localized to the R-U5 region of the HTLV-II 5' long terminal repeat and correlate with the region required for Rex function. The human immunodeficiency virus type 1 (HIV-1), has an analogous regulatory protein, Rev, which directly binds to and mediates its action through the Rev-responsive element located within the HIV-1 env gene. We demonstrated that HTLV-II Rex rescued an HIV-1JR-CSF Rev-deficient mutant, although inefficiently. This result is consistent with a weak binding activity to the HIV-1 Rev-responsive element under conditions in which it efficiently bound the HTLV-II long terminal repeat RNA.

Most detailed analyses of the human immunodeficiency virus type 1 (HIV-1) rev gene product have relied on transfection of subgenomic env constructs into cells in which amplification of the transfected DNA occurs. This was necessitated by difficulties in quantitating low-abundance HIV-1 mRNA species and in distinguishing different RNAs of similar sizes. We have modified the conventional polymerase chain reaction method for general use as an extremely sensitive procedure for quantitative analysis of RNA species. Using this method, we assessed the role of the HIV-1 rev gene in viral replication following mutagenesis of an infectious molecular clone, HIV-1JR-CSF. Following transfection of wild-type and mutant proviral constructs, we can specifically detect unspliced RNA and distinguish between the spliced tat-rev and nef mRNAs, which are not resolved by standard RNA analyses. Our results show that the rev protein of HIV-1JR-CSF simultaneously down regulates the expression of tat-rev and nef RNAs and up regulates the level of unspliced full-length HIV-1 RNA. A cis-acting element(s), located exclusively within the env sequences, is essential to exhibit this regulation. Fractionation of cells shows that the ultimate effect of Rev is to direct the appearance of unspliced or singly spliced RNAs in the cytoplasm. Models are discussed for possible mechanisms of Rev action.

The Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1) is required for protein expression from the HIV-1 RNAs which contain a binding site for the Rev protein, termed the Rev-responsive element (RRE). This transactivator acts both at the level of splicing/transport of nuclear RNAs and at the level of translation of cytoplasmic RNAs. We used a monoclonal antibody specific for the HIV-1 Rev protein to immunoprecipitate cellular extracts from HIV-1-infected and -transfected cells. High levels of specific binding of wild-type Rev to the RRE-containing RNAs were found in cytoplasmic, but not nuclear, extracts from these cells. A Rev mutant which lacked both nuclear and cytoplasmic Rev function but retained RNA binding in vivo was generated. This binding was detectable with both nuclear and cytoplasmic extracts. These results verify the existence of direct binding of Rev to HIV-1 RNAs in vivo and conclusively prove that binding of Rev is not sufficient for nuclear or cytoplasmic Rev function. The results also support a direct role for Rev in the nuclear export and translation of HIV-1 RNAs.

Only a fraction of retroviral primary transcripts are spliced to subgenomic mRNAs; the unspliced transcripts are transported to the cytoplasm for packaging into virions and for translation of the gag and pol genes. We identified cis-acting sequences within the gag gene of Rous sarcoma virus (RSV) which negatively regulate splicing in vivo. Mutations were generated downstream of the splice donor (base 397) in the intron of a proviral clone of RSV. Deletion of bases 708 to 800 or 874 to 987 resulted in a large increase in the level of spliced RSV RNA relative to unspliced RSV RNA. This negative regulator of splicing (nrs) also inhibited splicing of a heterologous splice donor and acceptor pair when inserted into the intron. The nrs element did not affect the level of spliced RNA by increasing the rate of transport of the unspliced RNA to the cytoplasm but interfered more directly with splicing. To investigate the possible role of gag proteins in splicing, we studied constructs carrying frameshift mutations in the gag gene. While these mutations, which caused premature termination of gag translation, did not affect the level of spliced RSV RNA, they resulted in a large decrease in the accumulation of unspliced RNA in the cytoplasm.

Background:Biallelic variants in POLR1Care associated with POLR3-related leukodystrophy (POLR3-HLD), or 4H leukodystrophy (Hypomyelination, Hypodontia, Hypogonadotropic Hypogonadism), and Treacher Collins syndrome (TCS). The clinical spectrum of POLR3-HLD caused by variants in this gene has not been described. Methods:A cross-sectional observational study involving 25 centers worldwide was conducted between 2016 and 2018. The clinical, radiologic and molecular features of 23 unreported and previously reported cases of POLR3-HLD caused by POLR1Cvariants were reviewed. Results:Most participants presented between birth and age 6 years with motor difficulties. Neurological deterioration was seen during childhood, suggesting a more severe phenotype than previously described. The dental, ocular and endocrine features often seen in POLR3-HLD were not invariably present. Five patients (22%) had a combination of hypomyelinating leukodystrophy and abnormal craniofacial development, including one individual with clear TCS features. Several cases did not exhibit all the typical radiologic characteristics of POLR3-HLD. A total of 29 different pathogenic variants in POLR1Cwere identified, including 13 new disease-causing variants. Conclusions:Based on the largest cohort of patients to date, these results suggest novel characteristics of POLR1C-related disorder, with a spectrum of clinical involvement characterized by hypomyelinating leukodystrophy with or without abnormal craniofacial development reminiscent of TCS.