Severe cryptococcal-associated neurological immune reconstitution inflammatory syndrome in a renal transplant recipient treated with adalimumab.

Cryptococcosis is a major concern in organ transplant recipients. A decrease in immunosuppressants following the initiation of antifungal therapy is currently recommended, but can occasionally be complicated by the onset of immune reconstitution inflammatory syndrome (IRIS). We report on a case of cryptococcosis in a kidney transplant recipient, compounded by severe neurological IRIS, the outcome of which was unfavorable despite the use of anti-tumor necrosis factor-alpha monoclonal antibodies.

Nerve growth factor (NGF) induces motoneuron apoptosis in rat embryonic spinal cord in vitro.

Recent studies have demonstrated that nerve growth factor (NGF) induces apoptosis of several cell types in the central nervous system through its low-affinity p75 neurotrophin receptor (p75NTR). To test the effect of NGF on embryonic motoneuron survival, we developed an organotypic culture system which allowed the in vitro development of intact embryonic rat spinal cords. In our system, neural tubes were taken and cultured at E13, just before the onset of physiological motoneuron death. After 2 days in vitro (DIV), motoneurons underwent apoptosis over a time-course similar to that in vivo. In this system, the addition of NGF (200 ng/mL) for 2 days enhanced the number of apoptotic motoneurons by 37%. This pro-apoptotic effect was completely reversed by the blocking anti-p75NTR (REX) antibody which inhibits NGF binding to p75NTR. Other neurotrophins, e.g. brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3) and neurotrophin 4/5 (NT4/5) did not have any effect, while glial cell-derived neurotrophic factor (GDNF) promoted motoneuron survival. Anti-BDNF blocking antibodies enhanced motoneuron death indicating that endogenous BDNF promotes motoneuron survival in explants. Our results demonstrate, for the first time, that NGF can induce embryonic motoneuron apoptosis through its receptor p75NTR.

Subcellular distribution of survival motor neuron (SMN) protein: possible involvement in nucleocytoplasmic and dendritic transport.

Spinal muscular atrophy (SMA) is among the most common recessive autosomal diseases and is characterized by the loss of spinal motor neurons. A gene termed 'Survival of Motor Neurons' (SMN) has been identified as the SMA-determining gene. Recent work indicates the involvement of the SMN protein and its associated protein SIP1 in spliceosomal snRNP biogenesis. However, the function of SMN remains unknown. Here, we have studied the subcellular localization of SMN in the rat spinal cord and more generally in the central nervous system (CNS), by light fluorescence and electron microscopy. SMN immunoreactivity (IR) was found in the different regions of the spinal cord but also in various regions of the CNS such as the brainstem, cerebellum, thalamus, cortex and hippocampus. In most neurons, we observed a speckled labelling of the cytoplasm and a discontinuous staining of the nuclear envelope. For some neurons (e.g. brainstem nuclei, dentate gyrus, cortex: layer V) and, in particular in motoneurons, SMN-IR was also present as prominent nuclear dot-like-structures. In these nuclear dots, SMN colocalized with SIP1 and with fibrillarin, a marker of coiled bodies. Ultrastructural studies in the anterior horn of the spinal cord confirmed the presence of SMN in the coiled bodies and also revealed the protein at the external side of nuclear pores complexes, in association with polyribosomes, and in dendrites, associated with microtubules. These localizations suggest that, in addition to its involvement in the spliceosome biogenesis, the SMN protein could also play a part in nucleocytoplasmic and dendritic transport.

Axonal targeting of agrin in cultured rat dorsal horn neurons.

Agrin, a synaptic basal lamina protein synthesized by motoneurons is involved in the aggregation of nicotinic acetylcholine receptors (nAchRs) at the neuromuscular junction. Agrin transcripts are broadly expressed in the central nervous system (CNS) including non-cholinergic regions. This wide distribution of agrin mRNAs raises the question of its function in these areas. To approach this question, we analysed the expression and cellular distribution of agrin in primary cultures of rat embryonic dorsal horn neurons. Polymerase chain reaction analysis demonstrated that the four agrin isoform (B0, B8, B11, B19) mRNAs are expressed as early as 4 days in vitro, before the formation of functional synaptic contacts. Western blots also showed that agrin-like proteins are secreted in conditioned medium from 7 days cultures. We analysed the subcellular distribution of agrin by double immunolabeling and fluorescence microscopy. We found that agrin is synthesized by almost all neurons and was present in the somata and in the axons but not in dendrites within the sensitivity of the detection. This intra-axonal localisation of agrin could only be seen after permeabilization. Furthermore, agrin immunoreactive axons were found adjacent to gephyrin, the postsynaptic glycine receptor-associated protein. Altogether, our results suggest that, as established at the neuromuscular junction, agrin may be involved in pre- to postsynaptic interactions in the central nervous system.

Expression of glycine receptor alpha subunits and gephyrin in cultured spinal neurons.

The inhibitory glycine receptor is a pentameric membrane protein composed of alpha and beta subunits. In the postsynaptic membrane, the glycine receptor and the copurifying peripheral membrane protein gephyrin are clustered underneath glycine-releasing nerve terminals. Here, we describe the expression of gephyrin and the neonatal and adult glycine receptor alpha subunit isoforms alpha1 and alpha2 during in vitro differentiation of rat spinal neurons. Analysis by immunoassays and the reverse transcriptase-polymerase chain reaction showed that gephyrin and alpha subunit mRNA and protein levels exhibited a marked increase from 1 to 5 days in vitro, i.e. prior to the formation of functional synaptic contacts. Using confocal and standard immunofluorescence, we determined the number of immunoreactive cells and the cellular localization of the alpha subunits and gephyrin. At 3 days in vitro, glycine receptor immunoreactivity revealed by the monoclonal antibody mAb4a was found in < 10% of cells and was mainly localized intracellularly; in contrast, gephyrin was detected in in vitro, gephyrin was essentially localized at the neuronal surface. At this stage, the number of glycine receptor-positive cells approached that of gephyrin-containing neurons (50%), and glycine receptor antigen was found both intracellularly and at the periphery of the cells. The antibody mAb2b, which binds exclusively to the alpha1 subunit, revealed aggregates at the surface of a few neurons. At 10 days in vitro, glycine receptor and gephyrin staining was localized in clusters at the periphery of the soma and the neurites. This quantitative analysis corroborates temporal differences in the cellular distribution of gephyrin and glycine receptor alpha subunits, the former being accumulated first at the neuronal surface.

The inhibitory neuronal glycine receptor.

Glycine is a major inhibitory neurotransmitter in the spinal cord and in the brain stem, where it acts by activating a chloride conductance. The postsynaptic glycine receptor has been purified and contains two transmembrane subunits of 48 kDa (alpha) and 58 kDa (beta), and a peripheral membrane protein of 93 kDa. cDNA sequencing of the alpha and beta subunits has revealed a common structural organization and a strong homology between these polypeptides and the nicotinic acetylcholine and GABAA receptor proteins. The glycine receptor exhibits a heterogeneity resulting from the existence of several alpha subtypes with distinct functional properties and different developmental expressions. When present in the central nervous system in situ, as well as in primary cultures of spinal cord neurons, these receptors are localized at the postsynaptic membrane adjacent to the presynaptic release sites, thus forming functional microdomains at the neuronal surface. This distribution raises the question of the formation and the maintenance of the heterogeneity of the somato-dendritic plasma membrane.

[Elements concerning the cellular organization of glycine receptors dependent of chlorine]. / Quelques éléments concernant l'organisation cellulaire du récepteur glycine dépendent du chlore.

Glycine is one of the main inhibitory neurotransmitter in the central nervous system of vertebrates where it acts by activating a chloride conductance. The distribution of glycine receptor at the neuronal surface was analysed by immunocytochemistry with monoclonal antibodies raised against the purified receptor. In the rat spinal cord as well as in other areas of the central nervous system, these receptors are localized at the postsynaptic membrane and are concentrated in front of the presynaptic release sites. Thus, they define functional microdomains at the plasma membrane. A similar organisation was observed in a motor command neuron: the Mauthner cell of Teleosts. Further, in this model, a quantitative analysis using confocal microscopy has established that the postsynaptic microdomains are arranged according to a somatodendritic gradient with the larger clusters at the tip of the dendrites. The use of primary cultures of rat or mouse spinal cord neurons has allowed to study the ontogenesis of the glycine receptor. We have shown that these receptors are, here also, organized in clusters present at the neuronal surface and that the formation of these aggregates occurs simultaneously to the establishment of synaptic contacts.

Cell division is required for expression of v-myc transforming properties in chicken embryonic neuroretina cells.

We previously reported that avian retroviruses carrying the v-myc oncogene alone fail to induce sustained proliferation and transformation of non-dividing chicken neuroretina (CNR) cells from 7-day-old embryos. However, v-myc is capable of transforming CNR cells which have been induced to multiply by the v-mil oncogene. These results suggest that entry into the cell cycle is required for the transformation of CNR cells by v-myc. To further assess the role of cell division, we investigated the transforming properties of v-myc in CNR cells conditionally induced to divide by the v-src gene or by modified culture conditions. We show that v-myc transforms CNR cells infected with Rous sarcoma virus mutants which induce cell proliferation in the absence of transformation. Expression of these transforming properties in CNR cells infected with temperature-sensitive v-src mutants depends on the continuous mitogenic activity of p60v-src. We also report that v-myc is able to transform CNR cells and to increase their growth potential under culture conditions which allow transient multiplication of uninfected cells. However, these v-myc-transformed cells rapidly cease to divide when returned to culture conditions that restrict the growth of normal cells. Taken together, these results indicate that transformation of CNR cells by the v-myc oncogene continuously depends on their ability to enter the cell cycle.

Molecular and biological properties of c-mil transducing retroviruses generated during passage of Rous-associated virus type 1 in chicken neuroretina cells.

IC1, IC2, and IC3 are novel c-mil transducing retroviruses generated during serial passaging of Rous-associated virus type 1 (RAV-1) in chicken embryo neuroretina cells. They were isolated by their ability to induce proliferation of these nondividing cells. IC2 and IC3 were generated during early passages of RAV-1 in neuroretina cells, whereas IC1 was isolated after six consecutive passages of virus supernatants. We sequenced the transduced genes and the mil-RAV-1 junctions of the three viruses. The 5' RAV-1-mil junction of IC2 and IC3 was formed by a splicing process between the RAV-1 leader sequence and exon 8 of the c-mil gene. The 5' end of IC1 resulted from homologous recombination between gag and mil sequences. Reconstitution experiments showed that serial passaging of IC2 in neuroretina cells also led to the formation of a gag-mil-containing retrovirus. Therefore, constitution of a U5-leader-delta c-mil-delta RAV-1-U3 virus represents early steps in c-mil transduction by RAV-1. This virus further recombined with RAV-1 to generate a gag-mil-containing virus. The three IC viruses transduced the serine/threonine kinase domain of the cellular gene. Hence, amino-terminal truncation is sufficient to activate the mitogenic property of c-mil. Comparison of the transforming properties of IC2 and IC1 showed that the transduced mil gene, expressed as a unique protein independent of gag sequences, was weakly transforming in avian cells. Acquisition of gag sequences by IC1 not only increased the rate of virus replication but also enhanced the transforming capacity of the virus.

Activation and transduction of c-mil sequences in chicken neuroretina cells induced to proliferate by infection with avian lymphomatosis virus.

We report that nondividing neuroretina cells from chicken embryos can be induced to proliferate following infection with Rous-associated virus type 1 (RAV-1), an avian lymphomatosis retrovirus lacking transforming genes. Multiplication of RAV-1-infected neuroretina cells is observed after a long latency period and takes place initially in a small number of cells. We also show that serial virus passaging onto fresh neuroretina cultures leads to the generation of novel mitogenic viruses containing the mil oncogene. DNA analysis indicated that RAV-1 is the only provirus detected in cells infected at virus passage 1, whereas neuroretina cells infected at subsequent virus passages harbor mil-containing proviruses. Three viruses, designated IC1, IC2, and IC3, were molecularly cloned. Restriction mapping indicated that in each virus, truncated c-mil sequences were inserted within different portions of the RAV-1 genome. In addition, IC1 and IC2 viruses have transduced novel sequences that belong to the 3' noncoding portion of the c-mil locus. All three viruses induce neuroretina cell multiplication and direct the synthesis of mil-specific proteins. Proliferation of neuroretina cells infected at passage 1 of RAV-1 was not associated with any detectable rearrangement of c-mil, when a v-mil probe was used. However, these cells expressed high levels of an aberrant 2.8-kilobase mRNA hybridizing to mil but not to a long terminal repeat probe. Therefore, transcriptional activation of a portion of c-mil could represent the initial events induced by RAV-1 infection and lead to retroviral transduction of activated c-mil sequences.

A novel oncogene related to c-mil is transduced in chicken neuroretina cells induced to proliferate by infection with an avian lymphomatosis virus.

Non-dividing neuroretina cells from chicken embryos are induced to proliferate after a long latency, following infection with Rous associated virus type 1, an avian retrovirus which does not carry a transforming gene. We have isolated from these proliferating cells an acutely mitogenic retrovirus, designated IC10, which contains a novel oncogene. Nucleotide sequencing showed that the IC10 virus has transduced 1101 nucleotides of cellular origin inserted between the gag and env genes of RAV-1. This oncogene, designated v-Rmil, is 70.1% homologous to v-mil. v-Rmil encodes a protein of 40,976 daltons sharing 83.8% homology with the catalytic domain of the v-mil protein. Divergence with the v-mil gene product is observed at the NH2- and COOH-terminal portions of the v-Rmil protein. Restriction analysis of normal chicken DNA indicated that v-Rmil is derived from a cellular gene distinct from c-mil. The c-Rmil gene is transcribed through a major mRNA, greater than 10 kb in length, that is detected at much higher levels in neuroretinas, as compared to other embryonic tissues.

Transformed and tumorigenic phenotypes induced by avian retroviruses containing the v-mil oncogene.

Avian retrovirus MH2 contains two oncogenes, v-mil and v-myc. We have previously shown that a spontaneous mutant of MH2 (PA200-MH2), expressing only the v-mil oncogene, is able to induce proliferation of quiescent neuroretina cells. In this study, we investigated the transforming and tumorigenic properties of v-mil. PA200 induced fibrosarcomas in about 60% of the injected chickens, whereas inoculation of MH2 resulted mainly in the appearance of kidney carcinomas. Analysis of several parameters of transformation showed that PA200, in contrast to MH2, induced only limited in vitro transformation of fibroblasts and neuroretina cells. These results suggest that v-myc is the major transforming and tumorigenic gene in MH2-infected cells. This low in vitro transforming capacity differentiates v-mil not only from other avian oncogenes, but also from the homologous murine v-raf gene.

Characterization of a MH2 mutant lacking the v-myc oncogene.

We have previously reported that a virus, MH2-PA200, lacking the ability to transform quail embryo cells, could be isolated from wild type (wt) MH2 stocks passaged on chicken neuroretina cells. We report here the molecular cloning and extensive characterization of this MH2-PA200 provirus. Molecularly cloned MH2-PA200 DNA was found to stimulate the growth of neuroretina cells by transfection assays and our results indicate that this recombinant virus was derived from the RAV-1 helper virus, in which v-mil and a small part of v-myc of MH2 were acquired at the expense of helper (delta gag-pol-delta env) sequences. In order to assess the precise boundary between the myc and env genes we determined the nucleotide sequence of the junction fragment and showed that 11 of 13 nucleotides of the env gene were identical to the myc sequence at the recombination point. The nucleotide sequence of the myc-env junction fragment of another similar and independently generated MH2 mutant showed similarly 9 nucleotides of homology between the env and myc sequences at the recombination point that took place at another site, suggesting that a homologous recombination occurred between MH2 and RAV-1 viruses to generate MH2-PA200 and similar mutants.

v-mil induces autocrine growth and enhanced tumorigenicity in v-myc-transformed avian macrophages.

MH2, an avian retrovirus containing the v-myc and v-mil oncogenes, rapidly transforms chick hematopoietic cells in vitro. The transformed cells belong to the macrophage lineage and proliferate in the absence of exogenous growth factors. Here we analyze a series of MH2 deletion mutants and show that these two oncogenes together establish an autocrine growth system in which v-myc stimulates cell proliferation, while v-mil induces the production of chicken myelomonocytic growth factor (cMGF). We also demonstrate that these two oncogenes cooperate in vivo. MH2 efficiently induces monocytic leukemias and liver tumors, while deletion mutants lacking either a functional v-mil or v-myc do not.

Characterization of a myc-containing retrovirus generated by propagation of an MH2 viral subgenomic RNA.

We have previously isolated, from wild-type MH2 virus that contains the two oncogenes mil and myc, mutants defective in one or the other oncogene product. We report here the molecular cloning and extensive characterization of MH2 CL25 provirus lacking the v-mil oncogene. Our results indicate that this virus corresponds to the propagation of the 2.8-kilobase subgenomic RNA of MH21.

Induction of proliferation or transformation of neuroretina cells by the mil and myc viral oncogenes.

The genome of the avian retrovirus MH2 contains, in addition to the v-myc oncogene shared with three other avian retroviruses (MC29, CMII and OK-10), a second cell-derived oncogene, v-mil (refs 1-3). Like the three other viruses, which contain only v-myc, MH2 induces mainly liver and kidney carcinomas in fowl and transforms fibroblasts and macrophages in vitro. However, MH2 and MC29 differ in their biological properties when assayed on cultures of chicken embryo neuroretina (NR) cells. Indeed, NR cells, which normally do not multiply in vitro, are induced to proliferate and become transformed upon infection with MH2, whereas infection with MC29 has no apparent effect on these cells. To analyse the functions of the two oncogenes of MH2, we isolated spontaneous and in vitro-constructed mutants of this virus and investigated their effects on NR cell multiplication and transformation. We report here that expression of v-mil is sufficient to induce NR cell proliferation, although it does not result in cell transformation. In addition, viruses expressing only the v-myc oncogene fail to induce any detectable change in NR cells. However, cooperation of the two oncogenes is required to achieve transformation of NR cells by MH2.