Biomedical subjects
Publications and source records attributed to D J Anderson.
In this article we present data from our laboratory, and review the literature available, on the potential association between HIV-1 and sperm. We focus on the use of PCR technology to answer this very important question, and emphasise the importance of using highly purified sperm preparations. We conclude that the likelihood of HIV infection/association with viable mature sperm is low.
The NEUROGENINS (NGNs) are neural-specific basic helix-loop-helix (bHLH) transcription factors. Mouse embryos lacking ngn1 fail to generate the proximal subset of cranial sensory neurons. ngn1 is required for the activation of a cascade of downstream bHLH factors, including NeuroD, MATH3, and NSCL1. ngn1 is expressed by placodal ectodermal cells and acts prior to neuroblast delamination. Moreover, NGN1 positively regulates the Delta homolog DLL1 and can be negatively regulated by Notch signaling. Thus, ngn1 functions similarly to the proneural genes in Drosophila. However, the initial pattern of ngn1 expression appears to be Notch independent. Taken together with the fact that ectopic ngn1 expression can convert ectodermal cells to neurons in Xenopus (Ma et al., 1996), these data and those of Fode et al. (1998 [this issue of Neuron]) identify ngns as vertebrate neuronal determination genes, analogous to myoD and myf5 in myogenesis.
The neuron-restrictive silencer factor NRSF (also known as REST and XBR) can silence transcription from neuronal promoters in non-neuronal cell lines, but its function during normal development is unknown. In mice, a targeted mutation of Rest, the gene encoding NRSF, caused derepression of neuron-specific tubulin in a subset of non-neural tissues and embryonic lethality. Mosaic inhibition of NRSF in chicken embryos, using a dominant-negative form of NRSF, also caused derepression of neuronal tubulin, as well as of several other neuronal target genes, in both non-neural tissues and central nervous system neuronal progenitors. These results indicate that NRSF is required to repress neuronal gene expression in vivo, in both extra-neural and undifferentiated neural tissue.
There is considerable evidence that the interleukin-1 (IL-1) system plays an important role in ovarian and testicular physiology, implantation, and other reproductive events. Human embryos express IL-1beta, IL-1 receptor type I (IL-1RtI), and IL-1 receptor antagonist (IL-1RA) at both the mRNA and protein levels. The presence of IL-1alpha and IL-1beta in oocyte-conditioned media and on the surface of human oocytes suggests that these cells may also produce this cytokine; however, whether the IL-1 system gene products are present as stable mRNAs in human gametes (oocytes and spermatozoa) has not yet been demonstrated. We used stringent cell separation techniques combined with reverse transcription-polymerase chain reaction to investigate the expression of various IL-1 system genes (IL-1alpha, IL-1beta, IL-1RtI, and IL-1RA) in human gametes and cumulus cells. Our results indicate that freshly isolated cumulus cells express all these IL-1 system components. On the other hand, IL-1alpha, IL-1beta, and IL-1RtI mRNAs were not found in either unfertilized or fertilized human oocytes, and a very few metaphase II human oocytes had transcripts for either secreted (10%) or intracellular (17%) IL-1RA. Mature spermatozoa did not contain mRNA for any of the of the IL-1 system components. The absence of informational RNA for the IL-1 system components in human unfertilized and polyploid oocytes and fresh immature oocytes suggests that maternal transcripts for these genes do not contribute to early embryo development. The presence of IL-1 components at the protein level in human oocytes may be due to binding of IL-1 produced by cumulus cells or other cell types, or to prior intrafollicle transcription and translation. Likewise, IL-1 system components do not appear to have a physiological role in mature spermatozoa since none of these components are present at the mRNA or protein levels, and important functional parameters such as motility and acrosome reaction appear not to be affected by IL-1beta in vitro. However, the abundant expression of IL-1alpha, IL-1beta, the IL-1RtI, and its antagonist IL-1RA by human cumulus cells provides further evidence that the IL-1 system plays a role in human ovarian physiology.
Vaccines are being sought for contraception and the prevention of sexually transmitted diseases. However, progress is slow in this area largely because of lack of information on induction of protective immune responses in genital tract mucosa. In this study, we investigated whether in vivo transfection with a model DNA-based antigen delivered by gene gun technology would induce an antibody response detectable in vaginal secretions. Female rats were immunized with plasmids encoding human growth hormone (HGH) under the control of a cytomegalovirus promoter (pCMV/HGH) via vaginal mucosa (V), Peyer's patch (PP), and/or abdominal skin (S) routes. Localization of HGH in the target tissues demonstrated that all three sites can be transfected in vivo with pCMV/HGH. Vaginal tissues expressed roughly the same level of plasmid as skin. Antibodies to HGH were detectable in serum and vaginal secretions in rats immunized with pCMV/HGH. In the rats primed and boosted vaginally, vaginal immunoglobulin A (IgA) and IgG antibody titers to HGH were sustained for at least 14 weeks, whereas rats immunized via other routes and protocols (S/V, S/S, PP/PP, or PP/V) did not consistently sustain significant vaginal antibody titers beyond week 6. DNA-based immunizations administered by the gene gun may be an effective method of inducing local immunity in the female genital tract.
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We have investigated the genetic circuitry underlying the determination of neuronal identity, using mammalian peripheral autonomic neurons as a model system. Previously, we showed that treatment of neural crest stem cells (NCSCs) with bone morphogenetic protein-2 (BMP-2) leads to an induction of MASH1 expression and consequent autonomic neuronal differentiation. We now show that BMP2 also induces expression of the paired homeodomain transcription factor Phox2a, and the GDNF/NTN signalling receptor tyrosine kinase c-RET. Constitutive expression of MASH1 in NCSCs from a retroviral vector, in the absence of exogenous BMP2, induces expression of both Phox2a and c-RET in a large fraction of infected colonies, and also promotes morphological neuronal differentiation and expression of pan-neuronal markers. In vivo, expression of Phox2a in autonomic ganglia is strongly reduced in Mash1 -/- embryos. These loss- and gain-of-function data suggest that MASH1 positively regulates expression of Phox2a, either directly or indirectly. Constitutive expression of Phox2a, by contrast to MASH1, fails to induce expression of neuronal markers or a neuronal morphology, but does induce expression of c-RET. These data suggest that MASH1 couples expression of pan-neuronal and subtype-specific components of autonomic neuronal identity, and support the general idea that identity is established by combining subprograms involving cascades of transcription factors, which specify distinct components of neuronal phenotype.
Recently, replication-deficient adenoviruses have received increasing attention as vector for gene delivery and as potential vaccine carriers. With the increased use of the vector in vivo and in clinical trails, the demand for a safe, rapid, and cost effective purification process has been heightened. In this report, a simple and efficient method for the purification of large quantities of live adenoviral vectors was developed. The process involved the replacement of cesium chloride (CsCl) gradients with sucrose gradients. Ultracentrifugation times were reduced and the desalting step eliminated, decreasing total preparation time by 15 hr. A 20-80% linear sucrose gradient provided optimal recovery of infectious viral particles and positioning of the viral band in the gradient. Purification with this gradient system produced a preparation containing 1.39 x 10(14) lac-forming units (lfu)/ml. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the process also removed all associated cellular proteins from the preparation. Studies have shown that direct lyophilization of the vector in sucrose after purification produces a product containing 1.4 x 10(12) lfu/ml. Minimal degradation was seen in the lyophilized preparation. A viral concentration of 6 x 10(11) lfu/ml was detected in the product after 150 days in storage at -20 degrees C. This approach will not only simplify the preparation of adenoviral vectors for in vivo studies and clinical trials, but will facilitate production of stable adenoviral formulations for oral gene delivery.
The discovery of (+/-)-epibatidine, a naturally occurring neuronal nicotinic acetylcholine receptor (nAChR) agonist with antinociceptive activity 200-fold more potent than that of morphine, has renewed interest in the potential role of nAChRs in pain processing. However, (+/-)-epibatidine has significant side-effect liabilities associated with potent activity at the ganglionic and neuromuscular junction nAChR subtypes which limit its potential as a clinical entity. ABT-594 [(R)-5-(2-azetidinylmethoxy)-2-chloropyridine] is a novel, potent cholinergic nAChR ligand with analgesic properties (see accompanying paper by Bannon et al., 1998b) that shows preferential selectivity for neuronal nAChRs and a consequently improved in vivo side-effect profile compared with (+/-)-epibatidine. ABT-594 is a potent inhibitor of the binding of [3H](-)-cytisine to alpha 4 beta 2 neuronal nAChRs (Ki = 37 pM, rat brain; Ki = 55 pM, transfected human receptor). At the alpha 1 beta 1 delta gamma neuromuscular nAChR labeled by [125I] alpha-bungarotoxin (alpha-Btx), ABT-594 has a Ki value of 10,000 nM resulting in a greater than 180,000-fold selectivity of the compound for the neuronal alpha 4 beta 2 nAChR. In contrast, (+/-)-epibatidine has Ki values of 70 pM and 2.7 nM at the alpha 4 beta 2 and alpha 1 beta 1 delta gamma nAChRs, respectively, giving a selectivity of only 38-fold. The S-enantiomer of ABT-594, A-98593 has activity at the neuronal alpha 4 beta 2 nAChR identical with ABT-594 (Ki = 34-39 pM), which demonstrates a lack of stereospecific binding similar to that reported previously for (+/-)-epibatidine. A similar lack of stereoselectivity is seen at the human alpha 7 receptor. However, A-98593 is 3-fold more potent at the neuromuscular nAChR (Ki = 3420 nM) and the brain alpha-Btx-sensitive nAChR (Ki = 4620 nM) than ABT-594. ABT-594 has weak affinity in binding assays for adrenoreceptor subtypes alpha-1B (Ki = 890 nM), alpha-2B (Ki = 597 nM) and alpha-2C (Ki = 342 nM), and it has negligible affinity (Ki > 1000 nM) for approximately 70 other receptors, enzyme and transporter binding sites. Functionally, ABT-594 is an agonist. At the transfected human alpha 4 beta 2 neuronal nAChR (K177 cells), with increased 86Rb+ efflux as a measure of cation efflux, ABT-594 had an EC50 value of 140 nM with an intrinsic activity (IA) compared with (-)-nicotine of 130%; at the nAChR subtype expressed in IMR-32 cells (sympathetic ganglion-like), an EC50 of 340 nM (IA = 126%); at the F11 dorsal root ganglion cell line (sensory ganglion-like), an EC50 of 1220 nM (IA = 71%); and via direct measurement of ion currents, an EC50 value of 56,000 nM (IA = 83%) at the human alpha 7 homooligimeric nAChR produced in oocytes. A-98593 is 2- to 3-fold more potent and displays approximately 50% greater intrinsic activity than ABT-594 in all four functional assays. In terms of potency, ABT-594 is 8- to 64-fold less active than (+/-)-epibatidine and also has less IA in these functional assays. ABT-594 (30 microM) inhibits the release of calcitonin gene-related peptide from C-fibers terminating in the dorsal horn of the spinal cord, an effect mediated via nAChRs. Pharmacologically, ABT-594 has an in vitro profile distinct from that of the prototypic nicotinic analgesic (+/-)-epibatidine, with the potential for substantially reduced side-effect liability and, as such, represents a potentially novel therapeutic approach to pain management.
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Molecular gradients have been postulated to control the topographic mapping of retinal axons in their central targets. Based initially on their expression patterns, and more recently on functional studies, members of the EphA subfamily of receptor tyrosine kinases and their ephrin-A ligands have been implicated in the guidance of retinal axons along the anterior-posterior axis of the chick optic tectum. The report that a receptor of the EphB subfamily, EphB2/Cek5/Nuk/Sek3, is expressed in a high ventral to low dorsal gradient in the developing chick retina and is present on ganglion cell axons suggests that it may be involved in the mapping of retinal axons along the corresponding dorsal-ventral axis of the tectum. To address this issue, we have determined the expression and distribution of ephrin-B1/LERK-2/Cek5-L and ephrin-B2/LERK-5/Htk-L/ELF-2, ligands for EphB2, in the developing chick retinotectal system using riboprobes, immunocytochemistry, and receptor affinity probes. Both ephrin-B1 and ephrin-B2 transcripts are expressed in a high dorsal to low ventral gradient in the developing retina, complementary to the distribution of EphB2. Ephrin-B1 and ephrin-B2 proteins are predominantly found in the developing plexiform layers, suggesting a role in the development of intraretinal connections. Neither protein is detected on ganglion cell axons. In tectum, ephrin-B1 transcripts are expressed in a high dorsal to low ventral gradient in the neuroepithelium and the protein is present along the processes of radial glia and is concentrated at their endfeet in the stratum opticum, at the time retinal axons are growing through it. This distribution of ephrin-B1 suggests that it influences retinal axon mapping along the dorsal-ventral tectal axis and may also be involved in intratectal development. In contrast, ephrin-B2 transcripts and protein are localized to the deeper retinorecipient laminae in the tectum at the time retinal axons begin to arborize in them, suggesting that this ligand may influence the laminar patterning of retinal axon terminations.
Growth factors can influence lineage determination of neural crest stem cells (NCSCs) in an instructive manner, in vitro. Because NCSCs are likely exposed to multiple signals in vivo, these findings raise the question of how stem cells would integrate such combined influences. Bone morphogenetic protein 2 (BMP2) promotes neuronal differentiation and glial growth factor 2 (GGF2) promotes glial differentiation; if NCSCs are exposed to saturating concentrations of both factors, BMP2 appears dominant. By contrast, if the cells are exposed to saturating concentrations of both BMP2 and transforming growth factor beta1 (which promotes smooth muscle differentiation), the two factors appear codominant. Sequential addition experiments indicate that NCSCs require 48-96 hrs in GGF2 before they commit to a glial fate, whereas the cells commit to a smooth muscle fate within 24 hr in transforming growth factor beta1. The delayed response to GGF2 does not reflect a lack of functional receptors; however, because the growth factor induces rapid mitogen-activated protein kinase phosphorylation in naive cells. Furthermore, GGF2 can attenuate induction of the neurogenic transcription factor mammalian achaete-scute homolog 1, by low doses of BMP2. This short-term antineurogenic influence of GGF2 is not sufficient for glial lineage commitment, however. These data imply that NCSCs exhibit cell-intrinsic biases in the timing and relative dosage sensitivity of their responses to instructive factors that influence the outcome of lineage decisions in the presence of multiple factors. The relative delay in glial lineage commitment, moreover, apparently reflects successive short-term and longer-term actions of GGF2. Such a delay may help to explain why glia normally differentiate after neurons, in vivo.
A quantitative enzyme-linked immunosorbent assay (ELISA) has been developed for the quantitation of HIV-1-specific immunoglobulins of the IgG, IgA, and IgM isotypes and was used to assess HIV-specific antibody concentrations in semen and cervicovaginal lavage (CVL) specimens. Immunoglobulin isotype concentrations were assessed by capture ELISA using immunoglobulin isotype-specific secondary antibodies and commercial IgG, IgA, and IgM standards. HIV-1 antibody detection kits (Abbott Laboratories, North Chicago, IL, U.S.A.) and immunoglobulin isotype-specific secondary antibodies were used to obtain optical density (OD) units for HIV-1-specific antibodies of each isotype. To determine the antibody concentrations from the OD values, ODs were compared with those from HIV-1-specific antibody isotype standards of known concentration obtained from CVL specimens of seropositive women by affinity binding to HIV antigen-coated beads and acid elution. The sensitivity of the HIV-1-specific immunoglobulin assay was 0.01 microg/ml for IgG, 0.04 microg/ml for IgA, and 0.08 microg/ml for IgM. The interassay coefficient of variation for the different immunoglobulin isotypes varied from 5% to 33%, being lowest for IgG and highest for IgM. HIV-1-specific antibodies were detected in all semen samples from seropositive men in concentrations ranging from 53 to 261 microg/ml. Thirteen of 14 samples contained high levels of HIV-1-specific IgG antibodies (22-72 microg/ml) whereas 10 of the 14 (71%) semen samples contained detectable but lower levels of HIV-1-specific IgA and IgM (maximum level: 3.7 microg/ml for IgA and 14.8 microg/ml for IgM). HIV-1-specific IgG antibodies were detected in all 196 CVL samples from seropositive women in concentrations ranging from 0.01 to 47 microg/ml, whereas only 16 women (8%) had IgA levels above the detectable limit (range, 1.4-3.9 microg/ml), and only eight women (4%) had IgM levels above the detectable limit (range, 1.8-8.6 microg/ml). None of 80 low-risk women or 20 low-risk men (negative controls) had detectable levels of HIV-1-specific antibodies in genital tract specimens. HIV-1-specific IgG levels in CVL specimens of seropositive women were significantly higher in individuals who had acquired HIV through heterosexual transmission, and a majority of women with elevated levels of HIV-specific IgA isotype antibodies in CVL samples had evidence of genital tract inflammation (>10[4] polymorphonuclear leukocytes [PMNs]/ml). Use of this quantitative method will facilitate direct comparison of data obtained within and among laboratories and enable further research on factors affecting antibody levels in genital tract secretions and their effects on HIV-1 transmission.
To isolate mouse neural crest stem cells, we have generated a rat monoclonal antibody to murine neurotrophin receptor (p75). We have immortalized p75+ murine neural crest cells by expression of v-myc, and have isolated several clonal cell lines. These lines can be maintained in an undifferentiated state, or induced to differentiate by changing the culture conditions. One of these cell lines, MONC-1, is capable of generating peripheral neurons, glia, and melanocytic cells. Importantly, most individual MONC-1 cells are multipotent when analyzed at clonal density. The neurons that differentiate under standard conditions have an autonomic-like phenotype, but under different conditions can express markers of other peripheral neuronal lineages. These lines therefore exhibit a similar differentiation potential as their normal counterparts. Furthermore, they can be genetically modified or generated from mice of different genetic backgrounds, providing a useful tool for molecular studies of neural crest development.
BACKGROUND: The interplay between growth factors and transcription factors in vertebrate neurogenesis is poorly understood. MASH1 is a basic helix-loop-helix (bHLH) transcription factor that is essential for autonomic neurogenesis. Bone morphogenetic protein (BMP) 2, and its relative BMP4, have been shown to induce expression of MASH1 and to promote autonomic neuronal differentiation in neural crest stem cells. The relationship between expression of MASH1 and the neurogenic competence of neural crest cells has not been investigated, however. RESULTS: We have examined the function of MASH1 in neurogenic competence using a population of immuno-isolated neural-crest-derived progenitor cells. Post-migratory neural crest cells isolated from fetal rat gut expressed Mash1, yet comprised a mixture of committed neuronal precursors and non-neuronal cells. The non-neuronal cells remained competent to differentiate to neurons, however, if challenged with BMP2. Such competence declines with time and is paralleled by a decline in Mash1 expression in the cells. Expression of endogenous Mash1 can be maintained by BMP2; in turn, constitutive expression of Mash1 from a retroviral vector maintains competence for neuronal differentiation in response to late addition of BMP2. CONCLUSIONS: These data suggest that MASH1 promotes competence for neurogenesis, in a manner similar to its homologs, the proneural genes achaete-scute in Drosophila. They also reveal an unexpected feedback interaction between BMP2 and MASH1 during neuronal differentiation. MASH1 may play multiple roles at successive stages of development within a neurogenic lineage, only one of which is revealed by a loss-of-function mutation.
Genetic studies in Drosophila and in vertebrates have implicated basic helix-loop-helix (bHLH) genes in neuronal fate determination and cell type specification. We have compared directly the expression of Mash1 and neurogenin1 (ngn1), two bHLH genes that are expressed specifically at early stages of neurogenesis. In the PNS these genes are expressed in complementary autonomic and sensory lineages. In the CNS in situ hybridization to serial sections and double-labeling experiments indicate that Mash1 and ngn1 are expressed in adjacent and nonoverlapping regions of the neuroepithelium that correspond to future functionally distinct areas of the brain. We also showed that in the PNS several other bHLH genes exhibit similar lineal restriction, as do ngn1 and Mash1, suggesting that complementary cascades of bHLH factors are involved in PNS development. Finally, we found that there is a close association between expression of ngn1 and Mash1 and that of two Notch ligands. These observations suggest a basic plan for vertebrate neurogenesis whereby regionalization of the neuroepithelium is followed by activation of a relatively small number of bHLH genes, which are used repeatedly in complementary domains to promote neural determination and differentiation.