Molecular fingerprinting reflects different histotypes and brain region in low grade gliomas.

BACKGROUND: Paediatric low-grade gliomas (LGGs) encompass a heterogeneous set of tumours of different histologies, site of lesion, age and gender distribution, growth potential, morphological features, tendency to progression and clinical course. Among LGGs, Pilocytic astrocytomas (PAs) are the most common central nervous system (CNS) tumours in children. They are typically well-circumscribed, classified as grade I by the World Health Organization (WHO), but recurrence or progressive disease occurs in about 10-20% of cases. Despite radiological and neuropathological features deemed as classic are acknowledged, PA may present a bewildering variety of microscopic features. Indeed, tumours containing both neoplastic ganglion and astrocytic cells occur at a lower frequency. METHODS: Gene expression profiling on 40 primary LGGs including PAs and mixed glial-neuronal tumours comprising gangliogliomas (GG) and desmoplastic infantile gangliogliomas (DIG) using Affymetrix array platform was performed. A biologically validated machine learning workflow for the identification of microarray-based gene signatures was devised. The method is based on a sparsity inducing regularization algorithm l1l2 that selects relevant variables and takes into account their correlation. The most significant genetic signatures emerging from gene-chip analysis were confirmed and validated by qPCR. RESULTS: We identified an expression signature composed by a biologically validated list of 15 genes, able to distinguish infratentorial from supratentorial LGGs. In addition, a specific molecular fingerprinting distinguishes the supratentorial PAs from those originating in the posterior fossa. Lastly, within supratentorial tumours, we also identified a gene expression pattern composed by neurogenesis, cell motility and cell growth genes which dichotomize mixed glial-neuronal tumours versus PAs. Our results reinforce previous observations about aberrant activation of the mitogen-activated protein kinase (MAPK) pathway in LGGs, but still point to an active involvement of TGF-beta signaling pathway in the PA development and pick out some hitherto unreported genes worthy of further investigation for the mixed glial-neuronal tumours. CONCLUSIONS: The identification of a brain region-specific gene signature suggests that LGGs, with similar pathological features but located at different sites, may be distinguishable on the basis of cancer genetics. Molecular fingerprinting seems to be able to better sub-classify such morphologically heterogeneous tumours and it is remarkable that mixed glial-neuronal tumours are strikingly separated from PAs.

Epidemiology of febrile neutropenia in children with central nervous system tumor: results from a single center prospective study.

Data regarding the epidemiology febrile neutropenia during chemotherapy for pediatric central nervous system neoplasia are scarce. Data retrieved from a prospective study performed from January 2002 to December 2004 at G.Gaslini Children Hospital, Genoa, Italy, where analyzed to evaluate proportions, rate for 1000 neutropenic days and etiology of fever in neutropenic children receiving gentle, standard, or peripheral blood stem cell transplant (PBSCT) therapy for central nervous system tumor. During the study duration, 243 periods of neutropenia (granulocyte count <1000/cmm), accounting for 3544 patient-days at risk, were documented in 62 children. A total of 72 febrile episodes were observed in 66 (27%) neutropenic periods, for a rate of 20.31. A primary febrile episode was observed in 10% of neutropenic periods after gentle chemotherapy, in 30% after standard chemotherapy, and in 48% after PBSCT (P<0.0001). The rate of primary febrile episodes was 6.19 after a gentle chemotherapy, 27.02 after standard treatment, and 31.02 after PBSCT (P<0.0001). In a multivariable regression model, the type of chemotherapy (gentle vs. standard and PBSCT) and the thresholds of granulocyte count at neutropenia onset (999-501/cmm and 500-101/cmm vs. ≤100/cmm) were the only factors significantly associated with the development of febrile neutropenia.

[Recent knowledge on the linkage of strain specific genotypes with clinical manifestations of human citomegalovirus disease]. / Attuali conoscenze sulla variabilità genetica del citomegalovirus umano e sua correlazione con il potenziale patogeno del virus: implicazioni diagnostico-terapeutiche.

Human citomegalovirus (CMV) is a beta-herpesvirus able to establish lifelong persistent infections which usually remain asymptomatic. However, severe diseases may develop in immunocompromised subjects (e.g., AIDS patients and transplant recipients) and if acquired in utero. Circulating CMV clinical strains display genetic polymorphisms in multiple genes, which may be implicated in CMV-induced immunopathogenesis, as well as strain-specific tissue-tropism, viral spread in the host cells and virulence, finally determining the wide spectrum of clinical manifestations of CMV disease. Current literature report a number of studies regarding the main CMV polymorphic genes (UL55-gB, UL144, UL73-gN, UL74-gO), their diagnostic and therapeutic impact, their potential clinical relevance as prognostic markers. This paper aims to critically analyse the results of these studies and evaluate the linkage of strain-specific genotypes with clinical manifestations of CMV disease and their perspective implications.

Cytomegalovirus gN genotypes distribution among congenitally infected newborns and their relationship with symptoms at birth and sequelae.

BACKGROUND: Cytomegalovirus (CMV) is the leading cause of congenital infection, with morbidity and mortality at birth and sequelae. Both host and viral factors may affect the outcome of infection. CMV strain virulence may depend on genetic variability in "key genes," such as UL73, which encodes the envelope glycoprotein gN. This study aimed to ascertain the role of gN variants as markers of pathogenicity and prognosis in newborns congenitally infected with CMV. METHODS: Seventy-four congenitally infected newborns were monitored for symptoms of CMV disease at birth and during long-term follow-up. The distribution of gN variants was analyzed in relation to virological parameters, clinical signs, laboratory and instrumental abnormalities at birth, and sequelae. Multivariate cluster analysis was used to test for differences in the distribution of variables. An independent validation cohort of the same size and modality of recruitment as the original population was examined by logistic regression to validate results. RESULTS: Univariate and cluster analyses suggest that newborns congenitally infected with CMV fall into 2 subpopulations on the basis of definite parameters of CMV disease. The first population with no symptoms at birth, negative instrumental findings, and a favorable long-term outcome was significantly associated with gN-1 and gN-3a genotypes. The second group with symptoms at birth, abnormal imaging results, and sequelae was associated with gN-4 genotypes (P<.05). The validation cohort further supports the results, indicating that genotypes gN-1 or gN-3a reduce the risk of sequelae 5 fold (95% confidence interval, 1.3-15.6 fold), whereas variants belonging to group gN-4 increase the risk of sequelae 8 fold (95% confidence interval, 2.6-25.8 fold). CONCLUSIONS: Results suggest that gN genotypes might be markers for virulence of CMV wild-type strains and a discriminating factor for selection of CMV-infected newborns who are at risk of developing sequelae.

Development of a multiplex PCR for the simultaneous amplification and genotyping of glycoprotein N among human cytomegalovirus strains.

Genomic variation among human cytomegalovirus (HCMV) strains is probably involved in HCMV-induced pathogenesis. The envelope glycoprotein N (gN) showed extensive genetic polymorphism as HCMV isolates have been clustered into four distinct gN variants (gN-1, gN-2, gN-3, gN-4) whose distribution has been analyzed worldwide using different methodological approaches (PCR-RFLP, PCR-Cloning, PCR-Sequencing). This paper describes a new method for concurrent detection of gN genotypes among HCMV strains using a multiplex gN-variants specific PCR plus visualization on agarose gel, avoiding subsequent steps such as cloning, restriction or sequencing. This novel approach will reduce costs and shorten the detection time of gN polymorphisms among HCMV clinical isolates.

Very low birth weight infants born to cytomegalovirus-seropositive mothers fed with their mother's milk: a prospective study.

OBJECTIVE: To assess the risk of post-natal cytomegalovirus (CMV) transmission to very low birth weight (VLBW) infants fed with their mother's fresh milk. STUDY DESIGN: Prospective, observational study of 80 VLBW infants and their 68 mothers. Infants' urine and their own mother's fresh breast milk were tested for CMV by means of culture tests once a week until discharge. CMV in infected milk and urine were genotyped. The clinical course, laboratory findings, and outcome of infants infected with CMV at 2 years of age are reported. RESULTS: Fifty-three mothers (78%) were CMV-seropositive at delivery. CMV was detected in the milk of 21 of 53 seropositive mothers (40%), and CMV was in the urine in 9 of 26 infants (35%) fed with CMV-positive milk. The same gN-genotype was found in milk and urine. Three infected infants <28 weeks gestational age (GA) had a mild sepsis-like illness. Five more infants had neutropenia, conjugated hyperbilirubinaemia, or both. Post-natal CMV infection occurred in 1 of 19 infants with a GA<28 weeks who were treated at birth with intravenous immunoglobulin versus 3 of 5 non-treated infants (P < .02). Symptomatic CMV infection was associated with bronchopulmonary dysplasia. No neurosensorial sequelae were found at 2 years of corrected age. CONCLUSIONS: CMV infection via fresh human milk is mild, self-limiting, and without sequelae. Very-low GA and pre-existing chronic diseases are associated with symptomatic infection.

Epidemiology of human cytomegalovirus strains through comparison of methodological approaches to explore gN variants.

Genomic variation among human cytomegalovirus (HCMV) wild-type strains is a well-documented phenomenon probably implicated in HCMV-induced immunopathogenesis. Extensive genetic polymorphism has been detected for the envelope glycoprotein N (gN) and HCMV clinical isolates have been clustered into seven distinct gN variants (gN-1, gN-2, gN-3a, gN-3b, gN-4a, gN-4b, gN-4c). Several studies from different research groups worldwide have addressed this topic using different methodological approaches (PCR-RFLP, PCR-Cloning, PCR-Sequencing) and sometimes yielding apparently conflicting results. This paper analyses the epidemiology of HCMV strains through analysis of gN variants, criticizing the methodological approaches and study populations by comparison of published reports.

Cytomegalovirus infection via mother's milk: could distinct virus strains determine different disease patterns in preterm twins?

We describe a case of cytomegalovirus infection via the mother's milk in preterm twins who showed a different disease pattern. Molecular analyses to genotype the viral genome disclosed a mixed population of virus strains in the milk. These isolates were differentially transmitted to each of the twins and this may be responsible of the different patterns of disease.

Testing a ratio of photosynthesis to O3 uptake as an index for assessing O3-induced foliar visible injury in poplar trees.

Visible foliar injury by ozone (ozone visible injury) is known as a biomarker to assess potential phytotoxicity of ozone. We investigated ozone visible injury in an ozone-sensitive poplar (Oxford clone) under a 2-year free-air controlled exposure (FACE) experiment and calculated three ozone indices (i.e., accumulative ozone exposure over 40 ppb during daylight hours (AOT40), phytotoxic ozone dose above a flux threshold of 0 nmol m-2 s-1 (POD0), and the cumulative value of the ratio of hourly ozone uptake to net photosynthesis (ΣU/P n ) to assess the critical level (CL) at the time of the first symptom onset of ozone visible injury. We tested the hypothesis that ozone injury depends both on the amount of ozone entering a leaf and on the capacity for biochemical detoxification or repair with photosynthesis as a proxy. The CLs at the time of the first symptom onset of ozone visible injury were 19 ppm h for AOT40, 26 mmol m-2 for POD0, and 1.2 mol mol-1 for ΣU/P n in Oxford clone at the ozone FACE experiment. Our findings were then verified by 4-year observation-based data in central Italy on Oxford clone and white poplar (Populus alba L.). These observation-based data indicated that we found ozone visible injury in Oxford clone even though AOT40 was relatively low (11.7 ppm h). On the other hand, when values of POD0 and ΣU/P n exceeded over the CLs, the occurrence of initial symptoms in Oxford clone was shown. White poplar did not show ozone visible injury. ΣU/P n of white poplar at the field sites reached ~1.0 mol mol-1 (less than the CL = 1.2 mol mol-1, which was obtained from O3 FACE) during May-September, although the values of POD0 were relatively high in white poplar (44-47 mmol m-2 during May-September). The result implies that ozone injury may have occurred in poplars when stomatal ozone flux exceeded the critical range of tolerance due to the assimilate shortage for repair and defense against ozone stress.

Monitoring for human cytomegalovirus infection in solid organ transplant recipients through antigenemia and glycoprotein N (gN) variants: evidence of correlation and potential prognostic value of gN genotypes.

Human cytomegalovirus (HCMV) ORF UL73 encodes the envelope glycoprotein gpUL73-gN, which shows seven genotypes (gN-1, gN-2, gN-3a, gN-3b, gN-4a, gN-4b, gN-4c). The goal of this study was to determine retrospectively the distribution of gN variants in solid organ transplant recipients with HCMV infection and to establish an association with parameters important for monitoring post-transplantation clinical course during a follow-up of up to 2 years. Peripheral blood leukocytes from 40 solid organ transplant recipients were analysed for pp65-antigen by immunofluorescence and gN genotyped by sequencing or RFLP analysis. A correlation between gN genotypes and antigenemia peak was found, showing a highly significant difference between gN-1 and gN-4b variants (P<0.005). In particular, gN-1 seems to be associated with patients developing low level antigenemia (<50 pp65-positive cells/2 x 10(5) PBLs; PPV = 90%), whereas gN-4b predicts significantly higher values (>50 pp65-positive cells/2 x 10(5) PBLs; PPV = 80%). Furthermore, the onset of positive antigenemia is significantly earlier in patients infected with a gN-4b strain, compared with those infected by a gN-1 variant. Reported data further support a role for gN genotypes in HCMV pathogenesis. gN-1 and gN-4b show a significantly different virulence and could serve as early predictors for the progression of HCMV infection in transplant patients.

Genomic variants among human cytomegalovirus (HCMV) clinical isolates: the glycoprotein n (gN) paradigm.

Human cytomegalovirus (HCMV) clinical isolates display genetic polymorphisms, which are supposed to be implicated in strain-specific tissue tropism and HCMV-induced immunopathogenesis. One highly variable gene is ORF UL73, encoding for the envelope glycoprotein gN, which displays both a structural and an immunologic role as a component of the high-molecular weight complex gC-II. UL73 showed clustered polymorphisms, which originate four distinct genomic variants, denoted gN-1, gN-2, gN-3, and gN-4. This review reports the main features of gN genotypes and their potential implications on HCMV biologic properties. The clinical impact of gN variants is also discussed. This overview on gN clustered polymorphisms should be useful as a prototype model for a better understanding of the biologic and clinical relevance of HCMV clinical isolates genetic variability.

Intrauterine cytomegalovirus infection and glycoprotein N (gN) genotypes.

BACKGROUND: Human cytomegalovirus (HCMV) clinical isolates display genetic polymorphisms, supposed to be related with strain-specific tissue-tropism and HCMV-induced immunopathogenesis. One recently discovered polymorphic gene is ORF UL73, encoding for the envelope glycoprotein gN. Among HCMV clinical strains, it shows four distinct genomic variants denoted as gN-1, gN-2, gN-3 and gN-4. OBJECTIVES: Aims of this study were to assess the prevalence of the different gN types in the populations examined and to investigate the possible relationship between genotypes and severity of congenital CMV disease. STUDY DESIGN: The gN genotyping was carried out by sequencing analysis of the HCMV ORF UL73. Comparisons were made by chi-square test and contingency tables. RESULTS: All the four gN genotypes can cause congenital infections and the overall distribution was as follows: gN-1, 23.6%; gN-2, 1.1%; gN-3, 12.9%; gN-4, 62.4%. None of them seems to be preferentially associated with vertical transmission or with acute outcome of congenital infection. However, considering the chronic outcome and long-term sequelae, there was a statistically significant (P<0.05) difference between congenitally infected infants with or without adverse chronic outcome. CONCLUSIONS: HCMV congenital infections, which displayed a prevalence of the gN-1 variants, seem to be associated with favorable chronic outcome.

Early classification of childhood focal idiopathic epilepsies: is it possible at the first seizure?

PURPOSES: To evaluate the possibility of early syndrome classification of idiopathic partial epilepsies in children at the first seizure. PATIENTS AND METHODS: In this observational study we prospectively evaluated 298 patients, aged between 1 month and 17 years and consecutively referred for the first unprovoked focal seizure. The whole cohort included 133 patients; the final analysis was carried out on 107 (59 males) individuals. Age at the first seizure ranged between 2.3 and 13.0 years. Clinical and EEG data of all patients were independently reviewed by two medical doctors. Patients were followed-up for at least 5 years, with a mean period of follow-up of 6.9 years. RESULTS: After the first seizure, a specific syndrome could be diagnosed in eighty (74.7%) children. In particular, Childhood Epilepsy with Centro-Temporal Spikes (CECTS) 42.9% of cases, Panayiotopoulos Syndrome (PS) 28.9%, idiopathic childhood occipital epilepsy of Gastaut (ICOE-G) 2.8%. Unclassified cases were 25.4%. At the end of the follow-up, the diagnosis was confirmed in 72 of 80 children (90%): BCECTS 89% of patients, PS 90% and ICOE-G 100%: among the unclassified cases, in 11 patients (40.7%) the diagnosis did not change, whereas 16 patients (59.3%) evolved into other syndromes or into atypical forms. CONCLUSIONS: At the onset an initial diagnosis is possible in the majority of cases; epilepsy syndromes can be identified at the time of the initial diagnosis and at follow up this diagnosis has not to be revised in 90% of the cases.

High levels of PROM1 (CD133) transcript are a potential predictor of poor prognosis in medulloblastoma.

The surface marker PROM1 is considered one of the most important markers of tumor-initiating cells, and its expression is believed to be an adverse prognostic factor in gliomas and in other malignancies. To date, to our knowledge, no specific studies of its expression in medulloblastoma series have been performed. The aims of our study were to evaluate the expression profile of the PROM1 gene in medulloblastoma and to assess its possible role as a prognostic factor. The PROM1 gene expression was evaluated by quantitative- polymerase chain reaction on 45 medulloblastoma samples by using specific dye-labeled probe systems. A significantly higher expression of PROM1 was found both in patients with poorer prognosis (P= .007) and in those with metastasis (P= .03). Kaplan-Meier analysis showed that both overall survival (OS) and progression-free survival (PFS) were shorter in patients with higher PROM1 mRNA levels than in patients with lower expression, even when the desmoplastic cases were excluded (P= .0004 and P= .002, for OS and PFS for all cases, respectively; P= .002 and P= .008 for OS and PFS for nondesmoplastic cases, respectively). Cox regression model demonstrated that PROM1 expression is an independent prognostic factor (hazard ratio, 4.56; P= .008). The result was validated on an independent cohort of 42 cases by microarray-based analysis (P= .019). This work suggests that high mRNA levels of PROM1 are associated with poor outcome in pediatric medulloblastoma. Furthermore, high PROM1 expression levels seem to increase the likelihood of metastases. Such results need to be confirmed in larger prospective series to possibly incorporate PROM1 gene expression into risk classification systems to be used in the clinical setting.

Remodelling of the nuclear lamina during human cytomegalovirus infection: role of the viral proteins pUL50 and pUL53.

A fundamental step in the efficient production of human cytomegalovirus (HCMV) progeny is viral egress from the nucleus to the cytoplasm of infected cells. In the family Herpesviridae, this process involves alteration of nuclear lamina components by two highly conserved proteins, whose homologues in HCMV are named pUL50 and pUL53. This study showed that HCMV infection induced the mislocalization of nuclear lamins and that pUL50 and pUL53 play a role in this event. At late stages of infection, both lamin A/C and lamin B showed an irregular distribution on the nuclear rim, coincident with areas of pUL53 accumulation. No variations in the total amount of nuclear lamins could be detected, supporting the view that HCMV induces a qualitative, rather than a quantitative, alteration of these cellular components, as has been suggested previously for other herpesviruses. Interestingly, pUL53, in the absence of other viral products, localized diffusely in the nucleus, whilst the co-expression and interaction of pUL53 with its partner, pUL50, restored its nuclear rim localization in distinct patches, thus indicating that pUL50 is sufficient to induce the localization of pUL53 observed during virus infection. Importantly, analysis of the nuclear lamina in the presence of pUL50-pUL53 complexes at the nuclear boundary and in the absence of other viral products showed that the two viral proteins were sufficient to promote alterations of lamins, strongly resembling those observed during HCMV infection. These results suggest that pUL50 and pUL53 may play an important role in the exit of virions from the nucleus by inducing structural modifications of the nuclear lamina.

Latency-associated human cytomegalovirus glycoprotein N genotypes in monocytes from healthy blood donors.

BACKGROUND: The beta-herpesvirus human cytomegalovirus (HCMV) infects a variety of cell types and maintains a lifelong relationship with its host by way of a latent infection in circulating monocytes, myeloid precursor cells, and the hematopoietic progenitor population. Viral strain heterogeneity, shown by gene polymorphisms, has been implicated in the majority of HCMV biologic behaviors. HCMV UL73 encodes the polymorphic envelope glycoprotein N (gN), which shows seven genotypes (gN-1, gN-2, gN-3a, gN-3b, gN-4a, gN-4b, and gN-4c). STUDY DESIGN AND METHODS: Monocyte subfractions from 64 HCMV-seropositive healthy blood donors were collected to analyze gN genotypes distribution in the few cells harboring the latent viral genome. Different experimental approaches to extract viral genomes from the monocyte population and amplify UL73 (polymerase chain reaction touchdown and nested) for subsequent genotyping were tested and compared with diagnostic gold standard. gN genotype distribution in monocytes from immunocompetent healthy carriers was compared with previously reported data obtained from patient populations with acute HCMV infections. RESULTS: The efficiency of UL73 amplification from monocytes of healthy seropositive blood donors was approximately 39 percent, one of the highest reported to date. The leading gN genotype was gN-1 (87%), whereas the gN-4 variant was poorly represented (13%). The comparison of gN genotypic frequencies in the immunocompetent healthy population with immunocompromised patients is discussed. CONCLUSIONS: This work further supports the idea that strain-specific features could determine the cell tropism and influence the onset of latency.

Genetic polymorphisms among human cytomegalovirus (HCMV) wild-type strains.

Human cytomegalovirus (HCMV) clinical isolates display genetic polymorphisms in multiple genes. Some authors have suggested that those polymorphisms may be implicated in HCMV-induced immunopathogenesis, as well as in strain-specific behaviours, such as tissue-tropism and ability to establish persistent or latent infections. This review summarises the features of the main clustered HCMV polymorphic open reading frames and also briefly cites other variable loci within the viral genome. The implications of gene polymorphisms are discussed in terms of potentially advantageous higher fitness obtained by the strain, but also taking into account that the published data are often speculative. The last section of this review summarises and critically analyses the main literature reports about the linkage of strain specific genotypes with clinical manifestations of HCMV disease in different patient populations affected by severe cytomegalovirus infections, namely immunocompromised subjects and congenitally infected newborns.

The tegument protein ppUL25 of human cytomegalovirus (CMV) is a major target antigen for the anti-CMV antibody response.

A viral protein of approximately 82 kDa is the only structural protein of human cytomegalovirus (CMV) that is strongly immunogenic during natural infection and the corresponding gene of which is still unknown. In this work, strong evidence is presented that this protein is the product of UL25.