Neurocognitive effects of methylphenidate on ADHD children with different DAT genotypes: a longitudinal open label trial.

The variable number of tandem repeat polymorphism in the 3'-untranslated region of the dopamine transporter gene (DAT) may influence the variability of the therapeutic response to methylphenidate (MPH) in Attention Deficit/Hyperactivity Disorder (ADHD). For this reason we evaluated the neuropsychological functioning after a prolonged period of MPH treatment and after a specific time from MPH suspension. Relationship between DAT VNTR genotypes and neurocognitive response to MPH was analyzed in a sample of 108 drug-naive ADHD patients. The performance of children with ADHD on measures of working memory, inhibition and planning was assessed at 4, 8 and 24 weeks and at 8 weeks after MPH withdrawal. Patients with 9/9 genotype evidenced an improvement in response inhibition and working memory only at 4 weeks of treatment, in planning at 24 weeks of therapy and after 8 weeks of MPH suspension. Patients with 9/10 showed an improvement in response inhibition at 4, 8 and 24 weeks of treatment, in planning at 24 weeks and after 8 weeks of MPH suspension. Patients with 10/10 evidenced an improvement in response inhibition and working memory at 4, 8 and 24 weeks of treatment and in planning at 4, 8 and 24 weeks of treatment and after 8 weeks of suspension. These results indicate that the 9/9 ADHD genotype has a different response at 24 weeks treatment with MPH. 10/10 DAT allele seems to be associated with an increased expression level of the dopamine transporter and seems to mediate the MPH treatment response in ADHD patients.

Interaction between catechol-O-methyltransferase (COMT) Val158Met genotype and genetic vulnerability to schizophrenia during explicit processing of aversive facial stimuli.

BACKGROUND: Emotion dysregulation is a key feature of schizophrenia, a brain disorder strongly associated with genetic risk and aberrant dopamine signalling. Dopamine is inactivated by catechol-O-methyltransferase (COMT), whose gene contains a functional polymorphism (COMT Val158Met) associated with differential activity of the enzyme and with brain physiology of emotion processing. The aim of the present study was to investigate whether genetic risk for schizophrenia and COMT Val158Met genotype interact on brain activity during implicit and explicit emotion processing. METHOD: A total of 25 patients with schizophrenia, 23 healthy siblings of patients and 24 comparison subjects genotyped for COMT Val158Met underwent functional magnetic resonance imaging during implicit and explicit processing of facial stimuli with negative emotional valence. RESULTS: We found a main effect of diagnosis in the right amygdala, with decreased activity in patients and siblings compared with control subjects. Furthermore, a genotype × diagnosis interaction was found in the left middle frontal gyrus, such that the effect of genetic risk for schizophrenia was evident in the context of the Val/Val genotype only, i.e. the phenotype of reduced activity was present especially in Val/Val patients and siblings. Finally, a complete inversion of the COMT effect between patients and healthy subjects was found in the left striatum during explicit processing. CONCLUSIONS: Overall, these results suggest complex interactions between genetically determined dopamine signalling and risk for schizophrenia on brain activity in the prefrontal cortex during emotion processing. On the other hand, the effects in the striatum may represent state-related epiphenomena of the disorder itself.

Catechol-O-methyltransferase Val(158)Met association with parahippocampal physiology during memory encoding in schizophrenia.

BACKGROUND: Catechol-O-methyltransferase (COMT) Val158Met has been associated with activity of the mesial temporal lobe during episodic memory and it may weakly increase risk for schizophrenia. However, how this variant affects parahippocampal and hippocampal physiology when dopamine transmission is perturbed is unclear. The aim of the present study was to compare the effects of the COMT Val158Met genotype on parahippocampal and hippocampal physiology during encoding of recognition memory in patients with schizophrenia and in healthy subjects. METHOD: Using blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI), we studied 28 patients with schizophrenia and 33 healthy subjects matched for a series of sociodemographic and genetic variables while they performed a recognition memory task. RESULTS: We found that healthy subjects had greater parahippocampal and hippocampal activity during memory encoding compared to patients with schizophrenia. We also found different activity of the parahippocampal region between healthy subjects and patients with schizophrenia as a function of the COMT genotype, in that the predicted COMT Met allele dose effect had an opposite direction in controls and patients. CONCLUSIONS: Our results demonstrate a COMT Val158Met genotype by diagnosis interaction in parahippocampal activity during memory encoding and may suggest that modulation of dopamine signaling interacts with other disease-related processes in determining the phenotype of parahippocampal physiology in schizophrenia.

A novel homozygous splice site mutation in the HPGD gene causes mild primary hypertrophic osteoarthropathy.

OBJECTIVES: Homozygous mutations in HPGD gene, encoding 15-hydroxyprostaglandin dehydrogenase, have recently been associated with primary hypertrophic osteoarthropathy (PHO). So far, only 7 HPGD alterations are known. In order to expand this mutational spectrum and better delineate the HPGD-related phenotype, we report the clinical and molecular characterisation of a 13-year-old boy and compare his features to known mutated patients. METHODS: The HPGD gene exons 1-7 and exon-intron junctions were analysed by direct sequencing. Previously published HPGD-mutated patients were systematically reviewed based on the original clinical description. RESULTS: A novel homozygous c.217+1G>A mutation affecting the obligatory donor splice site of HPGD exon 2 was identified in our proband who showed a mild form of PHO. Review of HPGD-mutated patients outlined all patients manifested digital clubbing, periostosis and acro-osteolysis. Hyperhidrosis (92%), arthralgia (65%) and eczema (33%) were variably associated features. Pachydermia (54%) was mild and mostly limited to palms and sole; cutis vertigis gyrata, blepharoptosis and severe skin thickening were never observed. Besides digital clubbing, PHO infants often presented patent ductus arteriosus (PDA) (32%) and delayed cranial sutures closure (55%). CONCLUSIONS: The present findings broaden the allelic spectrum of HPGD gene to include a novel c.217+1G>A mutation. Mutated patients display a homogeneous phenotype mainly consisting in digital clubbing, periostosis, acro-osteolysis, hyperhidrosis and mild pachydermia. Earliest manifestations include delayed closure of the cranial sutures and PDA. In conclusion, the information reported herein would facilitate the diagnosis of PHO due to HPGD mutations.

Deletions of NF1 gene and exons detected by multiplex ligation-dependent probe amplification.

To estimate the contribution of single and multi-exon NF1 gene copy-number changes to the NF1 mutation spectrum, we analysed a series of 201 Italian patients with neurofibromatosis type 1 (NF1). Of these, 138 had previously been found, using denaturing high-performance liquid chromatography or protein truncation test, to be heterozygous for intragenic NF1 point mutations/deletions/insertions, and were excluded from this analysis. The remaining 63 patients were analysed using multiplex ligation-dependent probe amplification (MLPA), which allows detection of deletions or duplications encompassing >or=1 NF1 exons, as well as entire gene deletions. MLPA results were validated using real-time quantitative PCR (qPCR) or fluorescent in situ hybridisation. MLPA screening followed by real-time qPCR detected a total of 23 deletions. Of these deletions, six were single exon, eight were multi-exon, and nine were of the entire NF1 gene. In our series, deletions encompassing >or=1 NF1 exons accounted for approximately 7% (14/201) of the NF1 gene mutation spectrum, suggesting that screening for these should now be systematically included in genetic testing of patients with NF1.

Pachydermoperiostosis: an update.

Pachydermoperiostosis (PDP) is a rare genodermatosis, characterized by pachydermia, digital clubbing, periostosis and an excess of affected males. Although an autosomal dominant model with incomplete penetrance and variable expression has been proved, both autosomal recessive and X-linked inheritance have been suggested. However, at present, genetic heterogeneity is not fully supported. The aim of this study is to review the clinical and pedigree data of 68 published PDP families, including 204 patients. This analysis has confirmed an autosomal dominant mutation in 37 families and suggested the existence of an autosomal recessive form in the remaining families. The two forms may differ in clinical severity, intrafamilial variability and prevalence of some features. Additionally, the marked skewed sex ratio could not be easily explained by an X-linked mutation, but alternative explanations (i.e. testosterone promoting proliferation) are discussed.

Metal complexes of lactoferrin and their effect on the intracellular multiplication of Legionella pneumophila.

The action of bovine lactoferrin saturated with iron, zinc and manganese on the intracellular multiplication of Legionella pneumophila in HeLa cells has been tested. The results obtained showed that lactoferrin did not influence the invasive efficiency of Legionella. The intracellular multiplication of the bacterium was inhibited by apo-lactoferrin and by lactoferrin saturated with manganese and zinc, whereas lactoferrin saturated with iron enhanced the intracellular growth. Experiments in parallel were performed with iron, manganese and zinc citrate to test the effect due to the metal ions alone. Even in this condition the addition of an iron chelate enhanced the multiplication of Legionella while the manganese chelate produced a certain inhibition.

Identification of a new control region in the genome of the DDP strain of BK virus isolated from PBMC.

The various strains of human polyomavirus BK (BKV) show a marked heterogeneity in the non-coding control region (NCCR), which includes the origin of replication and the regulatory region for early and late transcription. A new BKV strain (DDP, U91605) was identified by direct detection and sequencing of PCR products of BKV-NCCR DNA obtained from PBMC samples of HIV-positive or -negative subjects. The DDP strain NCCR sequence showed an organisation not described previously in vivo with the maximum homology with the archetypal strain (WW) (M34048), as compared with those collected in GenBank. Structurally, P68, Q39, and S68 boxes were perfectly conserved, whereas the R63 box was completely deleted. This deletion involves the loss of sequences able to bind cellular factors essential for the DNA transcription, such as NF1 binding sites, normally present twice in the R box and the modification of SP1. It is possible that these rearrangements represent a cause of the loss of the VP1 region observed in 9/22 PBMC samples and never observed in urine isolates, which are similar to the WW strain.

Transplacental transmission of human polyomavirus BK.

The presence of BK virus (BKV) and JC virus (JCV) in autopsy materials (placenta, brain, and kidney) of aborted fetuses was investigated by PCR using two sets of primers, specific for the regulatory region (RR) and for the capsid protein VP1, respectively. The RR of BKV was detected in 12 samples of placenta and brain and in nine samples of kidney obtained from 15 fetuses. Out of the 12 positive cases, four placentas, one brain, and three kidney samples also showed the presence of BKV DNA in the VP1 region. Of 12 placentas from a control group with a normal pregnancy outcome, the RR of BKV was detected in six samples, four of which were also positive for the VP1 region. None of the samples from either group was positive for the RR of JCV. In two cases, the nucleotide sequence of the BK RR demonstrated that the viruses isolated from maternal and fetal tissues showed a high homology with one another and had a characteristic deletion of the R63 box compared to the archetype strain. The results indicate that BKV may be transmitted vertically.

Multiplication of Legionella pneumophila in HeLa cells in the presence of cytoskeleton and metabolic inhibitors.

A study has been carried out on the action of cytoskeleton and metabolic inhibitors on intracellular multiplication in HeLa cells of a virulent strain of Legionella pneumophila serogroup 6. The effects of the substances were separately tested on both penetration and intracellular multiplication of L. pneumophila. Only cytochalasin A and 2-deoxy-D-glucose (2dG) affected bacterial internalisation, whereas intracellular multiplication was inhibited by cytochalasins A, B, C, D and J (D being the most active) and by 2dG with a dose-response effect. The action of 2dG was counteracted by 50 mM glucose. Experiments carried out with cytochalasin D and a rhodamine-phalloidin conjugate showed the involvement of cytoskeletal elements in intracellular multiplication of Legionella; compounds acting on microtubules had no effect.

Detection of BK polyomavirus genotypes in healthy and HIV-positive children.

Urine samples from 211 community children (3-7 years age), from 33 HIV type-1 infected children and from 56 HIV-negative children were collected and analyzed for the presence of BK virus (BKV) DNA by PCR. PCR amplifications were carried out using primers specific for the BKV structural region VP1. We also investigated the distribution of BKV subtypes by a restriction fragment polymorphism assay (RFLP). We demonstrated BKV DNA in 3.8% of 211 community children with a higher prevalence of subtype I. In HIV-1 positive children we detected BKV DNA in 2 urine samples (6%) out of 33, both belonging to subtype I. The HIV-negative cluster did not show any positivity to BKV DNA. The results confirm a more frequent primary BKV infection in children of 3-5 years of age and a higher prevalence in hospitalized children affected by HIV-1. The most relevant finding was that among both the community and HIV-1 positive children the subtype I was the most frequently detected.

Detection of JC and BK viral genome in specimens of HIV-1 infected subjects.

Human polyomaviruses JC and BK are ubiquitous in healthy human adults, persist as latent viruses and can be reactivated in the immunodeficient host giving different pathologies. Due to the experimental evidence of their potential oncogenicity and neurotropism, as well as to the enhanced viral production induced by co-infection with HIV-1, a possible role of these polyomaviruses has been suggested in AIDS-associated progressive multifocal leucoencephalopathy (PML) and Kaposi's sarcoma. JCV and BKV DNA was detected by PCR in urine and in peripheral blood mononuclear cells (PBMC) using primers specific for structural (VP1) and regulatory (R) regions. In HIV-positive subjects BKV and JCV sequences were found respectively in 8.1% and 31.6% of urine samples whereas in PBMC the positivity increased to 22.8% for JCV and in 51.1% for BKV. Our results indicated that, at DNA level, the presence of BKV and JCV in urine and PBMC was higher in HIV-1 positive subjects than in HIV-1 negative subjects and that, in contrast with JCV, BKV positivity was inversely related to blood CD4-level. Intravenous drug users (IVDU) showed significant increases in both BKV and JCV positivity, while an increased JCV viruria was found in homo-bisexuals compared to heterosexuals. The high prevalence of viral DNA in PBMC of both healthy and HIV-positive individuals agrees with the hypothesis that lymphocytes may represent a viral latency site permitting the establishment of virus persistence in affected organs, or a vehicle for the spread of the infection to different tissues.

Bafilomycin A1 and intracellular multiplication of Legionella pneumophila.

Multiplication of Legionella pneumophila in HeLa cells was found to be inhibited by noncytotoxic concentrations of bafilomycin A1, with blockage of bacterial growth at a concentration 15.6 nM. The inhibiting action was evident only when the antibiotic was present during the initial phase of intracellular multiplication, i.e., during the formation of the phagosome, whereas the addition of the drug did not affect microorganisms already actively multiplying within the phagosome.

The in vitro effect of monensin on BK polyomavirus replication.

The in vitro effect of monensin, a linear polyether, on the infection of Vero cells by BK polyomavirus, was investigated. Data reported in this paper showed an inhibition of BK viral replication by monensin as monitored by immunofluorescence and molecular hybridization. The inhibition of the synthesis of viral nuclear T antigen and the lack of production of viral mRNAs in monensin-treated cells suggest that the effect of this ionophore takes place at the level of the viral DNA delivery, by blocking the uncoating of BK virus or its transport to the nucleus.

Effect of monensin on the invasiveness and multiplication of Legionella pneumophila.

The polyether antibiotic monensin exhibited bacteriostatic activity against a clinical isolate of Legionella pneumophila in vitro. Experiments designed to test the effect of the compound on the invasiveness and multiplication of L. pneumophila in HeLa cells showed that, in the presence of the antibiotic, legionellas that penetrated the cells did not multiply. However, monensin did not alter the characteristics of phagosomes that contained ingested legionellas. In the presence of monensin, infected cells exhibited extensive vacuolation and a noticeable reduction in the number of intracellular micro-organisms was evident a few hours after infection.

Effect of various detergents on the interaction between BK virus and susceptible cells.

The activity of various types of detergents towards the infectivity and the hemagglutinating activity of BK virus was studied. Similarly to other non enveloped small viruses, BK virus showed a significant resistance to non cytotoxic concentrations of the compounds tested, with the exception of N-Dodecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate. In some cases, mainly with N-Octylglucoside, both the infectivity and the hemagglutinating activity of BK virus was enhanced.

Effect of sub-inhibitory concentrations of antibiotics on the hemolytic activity of Legionella.

The effect of sub-inhibitory concentrations of various antibiotics on the hemolytic activity of different strains of Legionella has been tested. By means of a gradient plate technique it was possible to demonstrate that in a limited range of sub-inhibitory concentrations, antibiotics did not affect bacterial growth but inhibited the hemolytic activity of the strains examined.