Virome definition in cerebrospinal fluid of patients with neurological complications after hematopoietic stem cell transplantation.

BACKGROUND: Neurological complications (NC) in allogeneic hematopoietic stem cell transplant (HSCT) recipients lead to long-term sequelae and result in significant morbidity and mortality. Since risk factors for NC include viral infection or reactivation, virome inspection after HSCT might be helpful to the clinical management of patients after HSCT. OBJECTIVES AND STUDY DESIGN: In this study we investigated whether any viruses are found in association with NC after HSCT. For this purpose, unbiased next generation sequencing (NGS) was used to characterize nucleic acid (NA) content in cerebrospinal fluid (CSF) taken at time of NC in 35 HSCT patients. Virome definition in CSF from non-transplanted subjects (controls) was also tested to define the commensal flora. RESULTS AND CONCLUSIONS: A higher number of reads/contigs mapped to viruses in patients compared to the controls (7,626 vs 235). Besides bacteriophages, Torque teno virus (TTV) was also identified in both controls and patients. Interestingly, a significantly higher number of TTV-like sequences was detected in the patient samples (7,236 vs 9), showing similarities to distinct genotypes; 3/2,575, 2/1,692 and 2/2,969 contigs/reads mapped to TTV11, TTV13 and Torque teno midi virus, respectively. In conclusion, unbiased NGS demonstrated to be a suitable approach to characterize the virome in samples containing limiting amounts of NA. The higher TTV levels and genetic diversity found in CSF of subjects with NC after HSCT might suggest a possible association between TTV reactivation and the disorder. However, further studies are needed to evaluate the possible role of TTV on NC in HSCT patients.

Virome characterisation from Guthrie cards in children who later developed acute lymphoblastic leukaemia.

BACKGROUND: Some childhood acute lymphoblastic leukaemias (ALL) can be traced back to a prenatal origin, where a virus infection could be involved in the first pre-leukaemic clone development. The DNA virome of 95 children who later developed ALL was characterised from neonatal blood spots (NBS) using unbiased next-generation sequencing (NGS) and compared with the virome of 95 non-ALL controls. METHODS: DNA was individually extracted from the ALL-patients and controls, pooled, randomly amplified and sequenced using the Illumina MiSeq Sequencing System. RESULTS: Virus-like sequences identified in both groups mapped to human endogenous retroviruses and propionibacterium phage, considered a part of the normal microbial flora. Potential pathogens human herpesvirus type 6 (HHV-6) and parvovirus B19 were also identified, but only few samples in both ALL and controls tested positive by PCR follow-up. CONCLUSIONS: Unbiased NGS was employed to search for DNA from potential infectious agents in neonatal samples of children who later developed ALL. Although several viral candidates were identified in the NBS samples, further investigation by PCR suggested that these viruses did not have a major role in ALL development.

Primary resistance to integrase strand-transfer inhibitors in Europe.

OBJECTIVES: The objective of this study was to define the natural genotypic variation of the HIV-1 integrase gene across Europe for epidemiological surveillance of integrase strand-transfer inhibitor (InSTI) resistance. METHODS: This was a multicentre, cross-sectional study within the European SPREAD HIV resistance surveillance programme. A representative set of 300 samples was selected from 1950 naive HIV-positive subjects newly diagnosed in 2006-07. The prevalence of InSTI resistance was evaluated using quality-controlled baseline population sequencing of integrase. Signature raltegravir, elvitegravir and dolutegravir resistance mutations were defined according to the IAS-USA 2014 list. In addition, all integrase substitutions relative to HXB2 were identified, including those with a Stanford HIVdb score ≥ 10 to at least one InSTI. To rule out circulation of minority InSTI-resistant HIV, 65 samples were selected for 454 integrase sequencing. RESULTS: For the population sequencing analysis, 278 samples were retrieved and successfully analysed. No signature resistance mutations to any of the InSTIs were detected. Eleven (4%) subjects had mutations at resistance-associated positions with an HIVdb score ≥ 10. Of the 56 samples successfully analysed with 454 sequencing, no InSTI signature mutations were detected, whereas integrase substitutions with an HIVdb score ≥ 10 were found in 8 (14.3%) individuals. CONCLUSIONS: No signature InSTI-resistant variants were circulating in Europe before the introduction of InSTIs. However, polymorphisms contributing to InSTI resistance were not rare. As InSTI use becomes more widespread, continuous surveillance of primary InSTI resistance is warranted. These data will be key to modelling the kinetics of InSTI resistance transmission in Europe in the coming years.

Neurotrophic actions of PACAP-38 and LIF on human neuroblastoma SH-SY5Y cells.

The neurotrophic actions of pituitary adenylate cyclase-activating polypeptide (PACAP)-38 and leukemia inhibitory factor (LIF) were investigated in human neuroblastoma SH-SY5Y cells. Effects on differentiation were assessed through monitoring morphological changes and Western blot analysis of the expression of neuronal marker proteins. In contrast to PACAP-38, which induced a 5.5-fold increase in the number of neurite-bearing cells, LIF had no significant effect on cell morphology compared to control cells over the 4-day time course. Cells co-treated with PACAP-38+LIF showed a similar increase in neurite-bearing cells compared to those treated with PACAP-38 alone. Cell morphology was similar for PACAP-38-treated and PACAP-38+LIF-co-treated cells, with the formation of bipolar neuron-like cells with long thin neurites, topped by growth cone-like structures and varicosities. SH-SY5Y cells express tyrosine hydroxylase (TH) but only low levels of the neuronal marker proteins: Bcl-2, GAP-43 and choline acetyltransferase (ChAT). Treatment of cells with PACAP-38 induced the expression of Bcl-2, GAP-43, and ChAT but did not appear to alter the expression of TH. LIF failed to induce the expression of GAP-43 and had little effect on the expression of TH, but did induce the expression of Bcl-2 and upregulated the expression of ChAT. Co-treatment with LIF had no effect on PACAP-38-induced expression of Bcl-2, GAP-43, and ChAT. Cells differentiated for 4 days with PACAP-38 or treated with LIF also displayed increased resistance to hypoxic conditions and to treatment with H2O2 and TNFalpha. The increased resistance to hypoxic conditions for PACAP-differentiated cells was blocked by the p38 MAP kinase inhibitor, SB203580, but not by the MEK1 inhibitor, PD98059. Additionally, cell proliferation assays show that LIF, but not PACAP-38, stimulates proliferation of SH-SY5Y cells, and this observed increase by LIF is not attenuated by co-treatment with PACAP. Further investigation of the intracellular signaling pathways mediating the neurotrophic effects of PACAP on SH-SY5Y cells indicate that neither phospholipase C activation nor Ca2+/calmodulin-dependent kinase II (CAMKII) are involved.

Effects of galanin receptor agonists on locus coeruleus neurons.

Galanin exerts an inhibitory effect on locus coeruleus (LC) neurons via a postsynaptic, as yet unidentified galanin receptor. Using an in vitro intracellular recording technique the effect of two galanin receptor agonists on LC neurons was investigated. Bath application of [Sar(1), D-Ala(12)]gal(1-16)-NH(2) (AR-M961), an agonist both at galanin R1 and R2 (GALR1, GALR2) receptors, evoked a reversible membrane hyperpolarization and inhibition of spike discharge in all LC neurons tested (n=42). The action of AR-M961 was blocked by tetraethylammonium chloride. Hyperpolarizing responses induced by AR-M961 were retained in the presence of tetrodotoxin and high Mg(2+)/low Ca(2+) media. The selective GALR2 agonist Gal(2-11)-NH(2) (AR-M1896) only caused inhibition of spike discharge and a slight hyperpolarization in 26 of 34 LC neurons tested, and was on a molar basis much weaker than AR-M961. These results suggest that it mainly is the GALR1 receptor that mediates hyperpolarization of LC neurons.

Receptor subtype-specific pronociceptive and analgesic actions of galanin in the spinal cord: selective actions via GalR1 and GalR2 receptors.

Galanin is a 29-aa neuropeptide with a complex role in pain processing. Several galanin receptor subtypes are present in dorsal root ganglia and spinal cord with a differential distribution. Here, we describe a generation of a specific galanin R2 (GalR2) agonist, AR-M1896, and its application in studies of a rat neuropathic pain model (Bennett). The results show that in normal rats mechanical and cold allodynia of the hindpaw are induced after intrathecal infusion of low-dose galanin (25 ng per 0.5 microl/h). The same effect is seen with equimolar doses of AR-M1896 or AR-M961, an agonist both at GalR1 and GalR2 receptors. In allodynic Bennett model rats, the mechanical threshold increased dose-dependently after intrathecal injection of a high dose of AR-M961, whereas no effect was observed in the control or AR-M1896 group. No effect of either of the two compounds was observed in nonallodynic Bennett model rats. These data indicate that a low dose of galanin has a nociceptive role at the spinal cord level mediated by GalR2 receptors, whereas the antiallodynic effect of high-dose galanin on neuropathic pain is mediated by the GalR1 receptors. Thus, a selective GalR1 agonist may be used to treat neuropathic pain.

The glycine residue in cyclic lactam analogues of galanin(1-16)-NH2 is important for stabilizing an N-terminal helix.

The neuropeptide galanin is a 29- or 30-residue peptide whose physiological functions are mediated by G-protein-coupled receptors. Galanin's agonist activity has been shown to be associated with the N-terminal sequence, galanin(1-16). Conformational investigations previously carried out on full-length galanin have, furthermore, indicated the presence of a helical conformation in the neuropeptide's N-terminal domain. Several cyclic lactam analogues of galanin(1-16)-NH2 were prepared in an attempt to stabilize an N-terminal helix in the peptide. Here we describe and compare the solution conformational properties of these analogues in the presence of SDS micelles as determined by NMR, CD, and fluorescence spectroscopy. Differences in CD spectral profiles were observed among the compounds that were studied. Both c[D4, K8]Gal(1-16)-NH2 and c[D4,K8]Gal(1-12)-NH2 adopted stable helical conformations in the micelle solution. On the basis of the analyses of their respective alpha H chemical shifts and NOE patterns, this helix was localized to the first 10 residues. The distance between the aromatic rings of Trp2 and Tyr9 in c[D4, K8]Gal(1-16)-NH2 was determined to be 10.8 +/- 3 A from fluorescence resonance energy transfer measurements. This interchromophore spacing was found to be more consistent with a helical structure than an extended one. Removal of the Gly1 residue in compounds c[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2 resulted in a loss of helical conformation and a concomitant reduction in binding potency at the GalR1 receptor but not at the GalR2 receptor. The nuclear Overhauser enhancements obtained for the Gly1 deficient analogues did, however, reveal the presence of nascent helical structures within the N-terminal sequence. Decreasing the ring structure size in c[D4, K8]Gal(1-16)-NH2 by replacing Lys8 with an ornithine residue or by changing the position of the single lysine residue from eight to seven was accompanied by a complete loss of helical structure and dramatically reduced receptor affinity. It is concluded from the data obtained for the series of cyclic galanin(1-16)-NH2 analogues that both the ring structure size and the presence of an N-terminal glycine residue are important for stabilizing an N-terminal helix in these compounds. However, although an N-terminal helix constitutes a predominant portion of the conformational ensemble for compounds c[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2, these peptides nevertheless are able to adopt other conformations in solution. Consequently, the correlation between the ability of the cyclic galanin analogues to adopt an N-terminal helix and bind to the GalR1 receptor may be considered as a working hypothesis.

The receptor for the orexigenic peptide melanin-concentrating hormone is a G-protein-coupled receptor.

Gene-knockout studies of melanin-concentrating hormone (MCH) and its effect on feeding and energy balance have firmly established MCH as an orexigenic (appetite-stimulating) peptide hormone. Here we identify MCH as the ligand for the orphan receptor SLC-1. The rat SLC-1 is activated by nanomolar concentrations of MCH and is coupled to the G protein G alpha i/o. The pattern of SLC-1 messenger RNA expression coincides with the distribution of MCH-containing nerve terminals and is consistent with the known central effects of MCH. Our identification of an MCH receptor could have implications for the development of new anti-obesity therapies.

The 1998 Wendell Hughes Lecture. Evisceration: is sympathetic ophthalmia a concern in the new millennium?

PURPOSE: To investigate a possible relationship between evisceration and sympathetic ophthalmia. METHODS: Data from Mt. Sinai Medical Center and University Hospitals of Cleveland were collected and histopathologic specimens were reviewed for 51 of 90 patients who underwent evisceration between 1980 and 1996 and who returned for follow-up examinations. Additionally, a survey was sent to members of the American Society of Ophthalmic Plastic and Reconstructive Surgery, the Uveitis Society, and the Eastern Ophthalmic Pathology Society to determine the number of enucleations and eviscerations performed and the documented incidence of sympathetic ophthalmia after evisceration. RESULTS: No clinical or histopathologic evidence of sympathetic ophthalmia after evisceration was found among patients treated at the two medical centers. The collective surveys showed a strong preference for enucleation over evisceration, but did not document evidence of sympathetic ophthalmia after evisceration. CONCLUSIONS: Evisceration is an effective and safe procedure with a low risk for sympathetic ophthalmia.

Cloning and characterization of a cDNA encoding a novel subtype of rat thyrotropin-releasing hormone receptor.

A cDNA encoding a thyrotropin-releasing hormone (TRH) receptor expressed in the pituitary was previously cloned (De La Pena, P., Delgado, L. M., Del Camino, D., and Barros, F. (1992) Biochem. J. 284, 891-899; De La Pena, P., Delgado, L. M., Del Camino, D., and Barros, F. (1992) J. Biol. Chem. 267, 25703-25708; Duthie, S. M., Taylor, P. L., Anderson, J., Cook, J., and Eidne, K. A. (1993) Mol. Cell Endocrinol. 95, R11-R15). We now describe the isolation of a rat cDNA encoding a novel subtype of TRH receptor (termed TRHR2) displaying an overall homology of 50% to the pituitary TRH receptor. Introduction of TRHR2 cDNA in HEK-293 cells resulted in expression of high affinity TRH binding with a different pharmacological profile than the pituitary TRH receptor. De novo expressed receptors were functional and resulted in stimulation of calcium transient as assessed by fluorometric imaging plate reader analysis. The message for TRHR2 was exclusive to central nervous system tissues as judged by Northern blot analysis. Studies of the expression of TRHR-2 message by in situ hybridization revealed a pattern of expression remarkably distinct (present in spinothalamic tract, spinal cord dorsal horn) from that of the pituitary TRH receptor (present in hypothalamus, and ventral horn of the spinal cord, anterior pituitary). Therefore, we have identified a novel, pharmacologically distinct receptor for thyrotropin-releasing hormone that appears to be more restricted to the central nervous system particularly to the sensory neurons of spinothalamic tract and spinal cord dorsal horn, which may account for the sensory antinociceptive actions of TRH.

The human 5-HT1A receptor: comparison of its binding properties in transfected cells and cortical tissue.

1. The binding characteristics of tritium labeled 8-hydroxy-dipropyl-aminotetralin, or [3H]8-OH-DPAT, to the serotonin1A (5-HT1A) receptor in the stably transfected HeLa cell clone HA6 and in human cortical tissue were examined and compared. 2. A series of kinetic studies of [3H]8-OH-DPAT binding to the transfected HA6 cell line demonstrated two components in both the association and the dissociation reactions. 3. In saturation experiments, at least two affinity states were unequivocally detected in the HA6 cell line and the human cortical tissue. Using isotopic dilutions, the binding isotherms were best fitted to a two-site model, and similar affinity values were obtained in both systems (KH approximately 1.1 nM and KL approximately 12-223 nM). 4. Most of the drugs used in competitions inhibited [3H]8-OH-DPAT binding, following a two-site model, and maintained their rank order of binding potency in both systems; that is, 5-HT > or = 8-OH-DPAT > buspirone > pindolol. Inconsistencies, however, were found for the antagonists NAN-190 and pindolol; only one inhibition constant was determined for HA6 cells, but two affinities were detected with cortical tissue. 5. The results indicate that, although data from binding studies using the cell expression system reflect, to a certain extent, those obtained with the cortical tissue, some discrepancies remained. 6. Finally, and in contrast with what is observed with the 5-HT1A receptor expressed in the HA6 cell line, it is possible that different receptors, or subtypes of one receptor, or even uptake sites normally expressed in cortical tissue, could interact with [3H]8-OH-DPAT or the competing drugs or both, thus leading to the observation of additional binding sites.

The human 5-hydroxytryptamine 1A receptor differentially modulates phospholipase C and adenylyl cyclase activities.

In this study, we quantitate and compare the ability of the 5-hydroxytryptamine 1A (5-HT1A) receptor to modulate the activities of phospholipase C and adenylyl cyclase as a function of receptor concentration. We used a single clonal cell line permanently expressing the human 5-HT1A receptor, and progressively depleted the receptor concentration using an alkylating antagonist (N8-bromoacetyl-N1-3'-(4-indolyloxy)-2'-hydroxypropyl-Z-1,8-diamin o-p-methane, (+-) Pindobind). For serotonin-induced phospholipase C stimulation, reductions in receptor number result in dose-response curves that shift downward and rightward, reflecting both a decreasing maximal effect as well as an increasing ED50. In contrast, depletion of more than 95% of the receptors has no effect on the maximal inhibition of forskolin-stimulated adenylyl cyclase activity. Moreover, at all receptor concentrations, the amount of serotonin required to produce half-maximal phospholipase C stimulation is several-fold more than that required to produce half-maximal inhibition of adenylyl cyclase activity. We conclude that the 5-HT1A receptor modulates these two pathways differently, and that the overall response to challenge with serotonin, in terms of both phosphatidyl inositol hydrolysis and cyclic AMP production, is dependent upon receptor number.