Quantification and characterization of granulocyte macrophage colony-stimulating factor activated human peripheral blood mononuclear cells by fluorine-19 cellular MRI in an immunocompromised mouse model.

PURPOSE: The purpose of this study was to test fluorine-19 (19F) cellular magnetic resonance (MRI) as a non-invasive imaging modality to track therapeutic cell migration as a surrogate marker of immunotherapeutic effectiveness. MATERIALS AND METHODS: Human peripheral blood mononuclear cell- (PBMC)-derived antigen presenting cell (APC) were labeled with a 19F-perfluorocarbon (PFC) and/or activated with granulocyte macrophage colony-stimulating factor (GM-CSF). Viability, phenotype and cell lineage characterization preceded 19F cellular MRI of PFC+ PBMC under both pre-clinical 9.4 Tesla (T) and clinical 3T conditions in a mouse model. RESULTS: A high proportion of PBMC incorporated PFC without affecting viability, phenotype or cell lineage composition. PFC+ PBMC were in vivo migration-competent to draining and downstream lymph nodes. GM-CSF addition to culture increased PBMC migration to, and persistence within, secondary lymphoid organs. CONCLUSION: 19F cellular MRI is a non-invasive imaging technique capable of detecting and quantifying in vivo cell migration in conjunction with an established APC-based immunotherapy model. 19F cellular MRI can function as a surrogate marker for assessing and improving upon the therapeutic benefit that this immunotherapy provides.

The effectiveness of the anti-CD11d treatment is reduced in rat models of spinal cord injury that produce significant levels of intraspinal hemorrhage.

We have previously reported that administration of a CD11d monoclonal antibody (mAb) improves recovery in a clip-compression model of SCI. In this model the CD11d mAb reduces the infiltration of activated leukocytes into the injured spinal cord (as indicated by reduced intraspinal MPO). However not all anti-inflammatory strategies have reported beneficial results, suggesting that success of the CD11d mAb treatment may depend on the type or severity of the injury. We therefore tested the CD11d mAb treatment in a rat hemi-contusion model of cervical SCI. In contrast to its effects in the clip-compression model, the CD11d mAb treatment did not improve forelimb function nor did it significantly reduce MPO levels in the hemi-contused cord. To determine if the disparate results using the CD11d mAb were due to the biomechanical nature of the cord injury (compression SCI versus contusion SCI) or to the spinal level of the injury (12th thoracic level versus cervical) we further evaluated the CD11d mAb treatment after a T12 contusion SCI. In contrast to the T12 clip compression SCI, the CD11d mAb treatment did not improve locomotor recovery or significantly reduce MPO levels after T12 contusion SCI. Lesion analyses revealed increased levels of hemorrhage after contusion SCI compared to clip-compression SCI. SCI that is accompanied by increased intraspinal hemorrhage would be predicted to be refractory to the CD11d mAb therapy as this approach targets leukocyte diapedesis through the intact vasculature. These results suggest that the disparate results of the anti-CD11d treatment in contusion and clip-compression models of SCI are due to the different pathophysiological mechanisms that dominate these two types of spinal cord injuries.

The NR1-4 C-terminus interferes with N-methyl-D-aspartate receptor-mediated excitotoxicity: evidence against a typical T/SXV-PDZ interaction.

The N-methyl-D-aspartate receptor (NMDAR) plays a key role in the neural plasticity that underlies learning and memory in vivo. The plasticity exhibited by NMDARs may also contribute to disease pathogenesis, as a number of disorders are caused or exacerbated by exaggerated NMDAR activity. The NMDAR is composed of two obligatory types of subunits, NR1 and NR2. These transmembrane proteins include large intracellular C-termini that have yet to be fully characterized. We have developed a three-color fluorescence system in order to visualize NMDAR expression in living cells. Using excitotoxicity as a proxy for exaggerated NMDAR activity, we analyzed the effect of over-expressing NR1-4 and NR2A C-terminal domains on exaggerated NMDAR function. We demonstrate that a determinant within the C-terminal domain of NR1-4 (C02') is important for NMDAR excitotoxicity, whereas no novel determinants were identified in the NR2A C-terminus. Through the use of heterologous cells, and by examining the interaction between the prototypical NMDAR-binding partner postsynaptic density-95 (PSD-95), we show that this effect is unlikely to be mediated through a classical interaction with PSD-95.

A monoclonal antibody to CD11d reduces the inflammatory infiltrate into the injured spinal cord: a potential neuroprotective treatment.

The accumulation of inflammatory cells in the lesion of a spinal cord injury (SCI) enhances secondary damage, resulting in further neurological impairment. High-dose methylprednisolone (MP) treatment is the only accepted treatment for inflammation secondary to human SCI but is minimally effective. Using a rat SCI model, we devised an anti-inflammatory treatment to block the infiltration of neutrophils and hematogenous monocyte/macrophages over the first 2 days postinjury by targeting the CD11dCD18 integrin. Anti-CD11d mAb administration following SCI effectively reduced neutrophil and macrophage infiltrate into lesions by 70% and 36%, respectively, over the first 72 h post-SCI. MP also reduced neutrophil and macrophage infiltrate by 60% and 28%, respectively, but by different mechanisms. The immunosuppression caused by anti-CD11d treatment was not sustained, as inflammatory cell numbers were not different from those observed in untreated SCI control animals at 7 days postinjury. In contrast, in MP-treated animals, the number of macrophages was still suppressed in the lesion while neutrophil numbers were significantly increased. These results suggest that anti-CD11d mAb treatment following SCI will minimize the destructive actions associated with early, uncontrolled leukocyte infiltration into the lesion while permitting the positive wound healing effects of macrophages at later time points.

The C-terminal C1 cassette of the N -methyl-D-aspartate receptor 1 subunit contains a bi-partite nuclear localization sequence.

The N -methyl-D-aspartate receptor (NMDAR) is a multimeric transmembrane protein composed of at least two subunits. One subunit, NR1, is derived from a single gene and can be subdivided into three regions: the N-terminal extracellular domain, the transmembrane regions, and the C-terminal intracellular domain. The N-terminal domain is responsible for Mg2+ metal ion binding and channel activity, while the transmembrane domains are important for ion channel formation. The intracellular C-terminal domain is involved in regulating receptor activity and subcellular localization. Our recent experiments indicated that the intracellular C-terminal domain, when expressed independently, localizes almost exclusively in the nucleus. An examination of the amino acid sequence reveals the presence of a putative nuclear localization sequence (NLS) in the C1 cassette of the NR1 intracellular C-terminus. Using an expression vector designed to test whether a putative NLS sequence is a valid, functional NLS, we have demonstrated that a bi-partite NLS does in fact exist within the NR1-1 C-terminus. Computer algorithms identified a putative helix-loop-helix motif that spanned the C0C1 cassettes of the C-terminus. These data suggest that the NR1 subunit may represent another member of a family of transmembrane proteins that undergo intramembrane proteolysis, releasing a cytosolic peptide that is actively translocated to the nucleus leading to alterations in gene regulation.

Distribution of an NMDA receptor:GFP fusion protein in sensory neurons is altered by a C-terminal construct.

The NMDA receptor plays an important role in mediating sensory input to the spinal cord. Domains within the C-terminus of the NMDA receptor bind to cytoskeletal proteins and facilitate membrane targeting and synaptic clustering, and may participate in regulation of receptor function. One strategy to manipulate NMDA receptor function is to express C-terminal constructs in neurons to disrupt synaptic clustering via competition for binding motifs in cytoskeletal proteins and postsynaptic densities. Biolistic particle-mediated gene transfer was used to deliver plasmid DNA into organotypic cultures of dorsal root ganglia (DRG). Fusion proteins consisting of recombinant (r)NMDA receptor subunit 1-1 (rNR1-1) deletion constructs and enhanced green fluorescent protein (GFP) were expressed in sensory neurons and demonstrated unique distribution patterns within the cell. Expression of the full length rNR1-1:GFP construct was cytosolic and localized to membranous patches similar to endogenous NR1-1 protein expression in sensory neurons. Expression of a construct containing only the C-terminus, GFP:C0C1C2, demonstrated nuclear and membranous localization. When the GFP:C0C1C2 construct was co-expressed with rNR1-1 in sensory neurons, membranous localization of rNR1-1 was disrupted. In contrast, co-expression of a C-terminal cassette lacking the C1 exon cassette, GFP:C0C2, with rNR1-1 did not alter the membranous distribution of rNR1-1. This observation verifies the utility of a gene transfer strategy to diminish membranous NR1-1 content by expressing a construct containing the C1 exon cassette.

In vivo magnetization transfer measurements of experimental spinal cord injury in the rat.

Magnetization transfer (MT) imaging techniques were implemented to study a clip compression model of spinal cord injury (SCI) in the rat. The purpose of this study was to determine if the magnetization transfer ratio (MTR) could be used to classify the stage and severity of SCI. Two clip compression injuries were studied: mild SCI and severe SCI. MTRs were determined for gray matter (GM) and white matter (WM) regions and the GM-WM contrast was determined on days 1 and 7 following surgery. Despite differences in pathologic features of mild and severe SCI, the GM-WM contrast did not allow discrimination between the two degrees of severity of SCI. WM MTR allowed differentiation of mild and severe SCI on day 1. These preliminary results suggest that WM MTR may provide an indication of the severity of injury in SCI. Magn Reson Med 45:159-163, 2001.

Inhibition of monocyte/macrophage migration to a spinal cord injury site by an antibody to the integrin alphaD: a potential new anti-inflammatory treatment.

The inflammatory response that ensues during the initial 48 to 72 h after spinal cord injury causes considerable secondary damage to neurons and glia. Infiltration of proinflammatory-activated neutrophils and monocytes/macrophages into the cord contributes to spinal cord injury-associated secondary damage. beta2 integrins play an essential role in leukocyte trafficking and activation and arbitrate cell-cell interactions during inflammation. The beta2 integrin, alphaDbeta2, is expressed on monocytes/macrophages and neutrophils and binds to vascular adhesion molecule-1 (VCAM-1). The increased expression of VCAM-1 during central nervous system (CNS) inflammation likely contributes to leukocyte extravasation into the CNS. Accordingly, blocking the interaction between alphaDbeta2 and VCAM-1 may attenuate the inflammatory response at the SCI site. We investigated whether the administration of monoclonal antibodies (mAbs) specific for the rat alphaD subunit would reduce the inflammatory response after a spinal cord transection injury in rats. At a 1 mg/kg dose two of three anti-alphaD mAbs caused a significant ( approximately 65%) reduction in the number of macrophages at the injury site and one anti-alphaD mAb led to a approximately 43% reduction in the number of neutrophils at the SCI site. Thus, our results support the concept that the alphaDbeta2 integrins play an important role in the trafficking of leukocytes to a site of central nervous system inflammation. This study also offers preliminary evidence that anti-alphaD mAbs can reduce the extravasation of macrophages and, to a lesser extent, neutrophils, to the SCI site.

Biolistic-mediated interleukin 4 gene transfer prevents the onset of type 1 diabetes.

We tested the efficacy of biolistic-mediated gene transfer as a noninvasive therapy for type 1 diabetes (T1D) in nonobese diabetic (NOD) mice by expression of murine interleukin 4 (mIL-4) cDNA. Epidermal delivery of 2 microg of DNA yielded transient detection of serum mIL-4, using a conventional cDNA expression vector. A vector stabilized by incorporation of the Epstein-Barr virus (EBV) EBNA1/oriP episomal maintenance replicon produced higher levels of serum mIL-4 that persisted for 12 days after inoculation. Although biolistic inoculation of either vector reduced insulitis and prevented diabetes, the protracted mIL-4 expression afforded by the EBV vector resulted in Th2-type responses in the periphery and pancreas and more significant protection from the onset of diabetes. Our studies demonstrate the efficacy of biolistic gene delivery of stabilized cytokine expression as a viable therapeutic approach to prevent the onset of T1D.

The HTLV-I orfI protein is recognized by serum antibodies from naturally infected humans and experimentally infected rabbits.

The mechanism of T-cell transformation by human T-cell lymphotropic virus type I (HTLV-I), though not completely understood, appears to involve the interactions of several viral and cellular proteins. One of these viral proteins, p12(I), encoded by HTLV-I orfI, is a weak oncogene that binds the 16-kDa subunit of the vacuolar ATPase and interacts with the immature beta and gamma(c) chains of the IL-2 receptor. We have expressed the singly spliced orfI cDNA in the baculovirus system and used the recombinant protein as a tool to assess the presence of antibodies in naturally or experimentally infected hosts. In addition, rabbit antisera were raised against various p12(I) synthetic peptides and used to identify three antigenic regions within p12(I), one between the two putative transmembrane regions of p12(I) and two at the carboxy-terminus of the protein. More importantly, sera from a naturally infected human (1 of 32) and experimentally infected rabbits (9 of 20) recognized the rp12(I), demonstrating orfI expression and immunogenicity in vivo. Taken together these data provide the first evidence of orfI expression during HTLV-I infections.

Herpes simplex viral and amplicon vector-mediated gene transfer into glia and neurons in organotypic spinal cord and dorsal root ganglion cultures.

The progression of neurodegenerative diseases and secondary consequences of spinal cord injury may be diminished by introducing transgenes to glia, spinal neurons, and/or sensory neurons. Organotypic cultures of spinal cord slices and dorsal root ganglia proved to be an excellent system in which to compare the relative neurotropism of a replication-defective recombinant herpes simplex virus and herpes virus-derived amplicon vectors. Hundreds of beta-galactosidase-expressing cells, transduced by the viral vectors, were observed in spinal cord slices 3 and 8 days postinfection. Immunostaining to identify the infected cell type indicated that oligodendrocytes were permissive for viral vector transduction of beta-galactosidase in the spinal cord slice, whereas neurons were not. Heparan sulfate proteoglycan, the initial receptor for herpes contact with cells, was highly expressed in the white matter of the spinal cord slice, but was negligible in the gray matter. In contrast to the spinal cord, many fewer cells were infected in the dorsal root ganglia (DRG) by these vectors, but a majority of infected cells were identified as sensory neurons. Heparan sulfate proteoglycan expression was abundant in the sensory fibers emanating from the DRG and also surrounded each neuron within the ganglion. Our results demonstrate HSV-induced transgene expression that is amenable to ex vivo assessment of its physiological impact.

HTLV type I/II in British Columbia Amerindians: a seroprevalence study and sequence characterization of an HTLV type IIa isolate.

It has been established that the human T cell lymphotropic viruses type I and II (HTLV-I and HTLV-II) are both present in some indigenous peoples of the Americas. While HTLV-I has been identified in coastal British Columbia Indians (BCIs), HTLV-II has not been previously reported in the BCIs or other Canadian Amerindians. The prevalence of HTLV-I and HTLV-II in these populations has not been extensively studied. In this article, we examine a group of BCIs from Vancouver Island who belong to the Nuu-Chah-Nulth and are known to have an increased incidence of rheumatic disease. In 494 serum samples from this tribe, the levels of prevalence of HTLV-I and HTLV-II were 2.8 and 1.6%, respectively. No association could be made between arthropathy and HTLV-I infection. In addition, we characterized an HTLV-II isolate of a BCI from the coastal mainland of British Columbia and with a history of intravenous drug abuse. This case represents the first molecular characterization of a Canadian Amerindian HTLV-II isolate: a subtype IIa virus with phylogenetic affinity for intravenous drug user isolates and containing an extended form of the Tax protein. These results are consistent either with this strain having been sampled from a polymorphic ancestral pool of HTLV-II that gave rise to the current epidemic spread of this virus by intravenous drug use and sexual transmission, or with its being "back-transmitted" into the BC Amerindian population in association with intravenous drug use.

A multi-mutant herpes simplex virus vector has minimal cytotoxic effects on the distribution of filamentous actin, alpha-actinin 2 and a glutamate receptor in differentiated PC12 cells.

To develop effective gene therapy techniques that target populations of neurons in the spinal cord, suitable vectors must be developed that will undergo efficient, retrograde transport from an appropriate peripheral site and will not be cytotoxic. Our previous work (LeVatte et al, 1998a) has demonstrated that a replication defective herpes simplex virus vector 14Hdelta3vhsZ, that has been substantially detoxified, is retrogradely transported from peripheral sites and can infect large numbers of the targeted spinal neurons. We plan to develop targeted gene therapy approaches designed to modulate the excitatory glutamatergic methyl-D-aspartate (NMDA) receptor in spinal cord neurons as a means of ameliorating a form of episodic high blood pressure that occurs after spinal cord injury. In this report, we demonstrate that, in differentiated PC12 cells, a neuronal-like cell line, the virus vector does not appear to alter aspects of the cytoskeletal architecture important to the proper distribution of the NMDA receptor. In turn, the distribution of endogenous NMDA receptor 1 subunit protein (NMDAR1) or a transfected NMDAR1-green fluorescent fusion protein was also found to be unaltered after vector infection. However, whereas endogenous NMDAR1 distribution was maintained, vector infection did tend to reduce the level of its expression. This drop in endogenous NMDAR1 expression coincided with the expression of the HSV immediate early genes ICP0 and ICP27 over the first 24-48 h. These results indicate that the 14Hdelta3vhsZ herpes simplex virus vector is suitable to use in future strategies to alter the level of gene expression in targeted populations of spinal cord neurons.

An apparent case of human T-cell lymphotropic virus type II (HTLV-II)-associated neurological disease: a clinical, molecular, and phylogenetic characterisation.

Several studies have reported an association between HTLV-II and a neurological condition which has come to be called HTLV-II-associated myelopathy and is similar, in some cases, to HTLV-I-associated myelopathy. To further explore the establishment of an etiological link between this virus and neurological disease, we determined the HTLV status of three individuals, one of which presented with symptoms of progressive ataxia. Since the patient with neurological disease and her husband were HTLV-II positive, we had the potential to study one of few cases of an HTLV-II-associated neurological disorder, and the first case in Canada. However, although the individual with the neurological disease was HTLV-II positive, we discovered that her brother, who displays the same clinical symptoms, was not positive for either HTLV-II or HTLV-I. Thus, disease association with HTLV-II became unsupportable. We present here, nevertheless, the first sequence and phylogenetic analysis of an HTLV-II isolate in Canada. This study suggests that cases of HTLV-II and neurological disease must be carefully investigated before any etiological conclusions can be made.

Cyclosporin A reduces the inflammatory response to a multi-mutant herpes simplex virus type-1 leading to improved transgene expression in sympathetic preganglionic neurons in hamsters.

Herpes simplex virus type 1 (HSV-1) based vectors hold great promise for gene transfer to CNS neurons. Problems such as loss of transgene expression, vector-associated cytotoxicity and the immune response to the vector or encoded transgene still remain obstacles to success. We used a replication-defective, HSV-1 vector (14Hdelta3vhsZ) that was engineered to have reduced cytotoxicity and express recombinant beta-galactosidase. A previous study in our laboratory showed no evidence for cytotoxicity in infected neurons although an inflammatory infiltrate occurred around infected cells and transgene expression was lost between 5 and 8 days. The immune response consisted of a primary response at the site of inoculation (adrenal gland), and a secondary immune response in the spinal cord around infected adrenal sympathetic preganglionic neurons due to retrograde transport of the vector. We tested whether conventional immunosuppressants could reduce the secondary immune response, leading to improved transgene expression at the secondary CNS site. 14Hdelta3vhsZ was injected into the adrenal gland in hamsters 1 day after immunosuppressant treatment began. Non-drug treated, 14Hdelta3vhzZ-infected hamsters were used as controls. Cyclosporin A administration led to the most persistent beta-galactosidase activity in neurons at 5 and 8 days. Methylprednisolone treatment resulted in the greatest reduction in the inflammatory cell infiltrate but the numbers of infected neurons did not increase concomitantly. This suggested no direct relationship between extent of the inflammatory cell infiltrate and level of transgene expression. These data demonstrate the potential of cyclosporin A as an immunosuppressant adjunct treatment for HSV-1 vector-mediated gene transfer from a peripheral site to neurons in the spinal cord.

Engineering of noninfectious HIV-1-like particles containing mutant gp41 glycoproteins as vaccine candidates that allow vaccinees to be distinguished from HIV-1 infectees.

Many AIDS vaccine candidates under development may elicit immune responses similar to those observed in and used to screen human immunodeficiency virus type 1 (HIV-1)-infected individuals. Therefore, it is important to develop vaccine candidates that incorporate antigenic markers and allow vaccinees to be distinguished from HIV-1 infectees. To this end, we introduced a series of mutations into and in the vicinity of the major immunodominant region (MIR) of gp41 (residues 598-609), a domain recognized by almost all HIV-1 infectees, and evaluated whether HIV-1-like particles incorporating such mutant glycoproteins could be expressed in mammalian cells. Results indicated that although up to three consecutive amino acids could be replaced within MIR without significantly affecting particle formation or gp160 processing, deletions within MIR impaired envelope processing. Replacement of HIV-1 MIR by part or most of the corresponding domain from other lentiviruses markedly decreased or abolished gp160 processing. Synthetic peptides corresponding to a mutated MIR incorporating three amino acid replacements were not recognized by a panel of sera from HIV-1 infectees, suggesting that HIV-1-like particles with this type of mutation represent potential candidate vaccines that could allow vaccinees to be distinguished from HIV-1 infectees.

Recombinant vaccinia viruses and gene gun vectors expressing the large subunit of Schistosoma mansoni calpain used in a murine immunization-challenge model.

Schistosomiasis is a parasitic disease affecting over 200 million people every year in tropical regions of the world. Drug treatment and other existing control measures are costly and have failed to eliminate the incidence of infection, morbidity and mortality due to Schistosoma mansoni infection. Vaccination of susceptible individuals using recombinant vaccines encoding key S. mansoni antigens may be the most effective and least expensive means of controlling schistosomiasis. A candidate vaccine antigen is p80, the large subunit of the S. mansoni protease, calpain. In our vaccine studies, we have employed both the wild-type p80 and a mutant p80 (mut p80) in which an active site amino acid was genetically altered to create a less proteolytically-active enzyme. Two vaccine delivery approaches were implemented using p80 or mut p80 as vaccine antigen: recombinant vaccinia virus (RVV) inoculation and DNA immunization via the Accell gene gun (GG) delivery system. RVV's expressing p80 and mut p80 were generated and tested for recombinant protein expression in vitro. These RVV's were tested for protective capacity in mouse challenge studies. Neither subcutaneous nor intranasal vaccinations with RVV-p80 or RVV-mut p80 were capable of significantly reducing the mean worm burdens of vaccinated mice. A GG-RVV combination immunization regime using WRG-vectors encoding p80 and mut p80 for GG priming and the RVV's for boosting prior to S. mansoni challenge infection was performed and no significant protection was obtained over two repeated studies. However, duplicate challenge studies involving GG immunization of mice with WRG-vectors encoding p80 or mut p80 revealed that 3 inoculations of mice with WRG-full5' mut p80 (containing the full 5' untranslated region of mut p80) provided 60% protection which was statistically significant (p < 0.05). These preliminary in vivo studies demonstrate the potential for further study of the protection afforded by gene gun-delivered WRG-full5' mut p80 into subsequently-challenged mice. Such studies may pave the way to effective vaccination of humans using WRG DNA vectors expressing a schistosomal calcium-activated neutral protease.

Analysis of a multi-mutant herpes simplex virus type 1 for gene transfer into sympathetic preganglionic neurons and a comparison to adenovirus vectors.

A non-replicating triple-mutant herpes simplex virus (14H delta 3vhsZ) expressing the bacterial marker enzyme beta-galactosidase, was assessed for neurotropism and cytopathic effects as a vector for gene transfer into differentiated phaeochromocytoma 12 cells in vitro and into spinal sympathetic neurons in vivo. In the in vivo study, the 14H delta 3vhsZ was injected into the adrenal gland of hamsters. For comparison, an evaluation of two adenovirus vectors, AdCA17lacZ and AdCA36lacZ, was performed. Infection of the differentiated phaeochromocytoma 12 cells by 14H delta 3vhsZ resulted in intense beta-galactosidase staining in 80-90% of the cells without changes in cell morphology, detected by light microscopy, after a period of four days. No cytoskeletal disruption was detected by immunocytochemistry for the neurofilament protein and no apoptosis was demonstrated by the Hoescht stain for nuclear chromatin in virus-infected cells in comparison to mock-infected control cells. Twoto three days after adrenal inoculation with 14H delta 3vhsZ, beta-galactosidase was detected in 240 preganglionic neurons per hamster (n = 8), a number equal to about 25% of the population of targeted neurons. The beta-galactosidase reaction product extended throughout the normal kite-shaped neuronal somata and extensive dendritic arbour. The number decreased to 120 by five days (n = 3) and to two by eight days (n = 4). This decrease was presumably due to loss of expression of the marker gene and not to cell death because, at eight days, the number of sympathetic pregnanglionic neurons in the nucleus intermediolateralis, pars principalis, that were immunoreactive for the neurotransmitter enzyme choline acetyltransferase, and demonstrated nicotinamide adenine dinucleotide phosphate-diaphorase activity, were the same on the infected left side of the cord as on the uninfected right side. Inflammatory cells surrounded some of the infected neurons at five days but by eight days the infiltrate was reduced. Infection of differentiated phaeochromocytoma 12 cells by AdCA17lacZ and AdCA36lacZ also resulted in marker gene expression in a large proportion of the cells (80-90%) in the absence of cytopathic effects. In contrast, four days after adrenal injection of AdCA17lacZ or AdCA36lacZ (n = 5 for each) only an average of three preganglionic neurons per hamster expressed beta-galactosidase activity, despite clear adrenal infection. AdCA17lacZ and AdCA36lacZ both produced light patches of staining confined to the neuronal soma. These neurons had normal morphology but sometimes were surrounded by an inflammatory infiltrate. In conclusion, the non-replicating herpes simplex virus, 14H delta 3vhsZ, had minimal cytotoxic effects in neurons, in vitro or in vivo, and was efficiently transported from the adrenal gland to infect many sympathoadrenal pregnanglionic neurons. In contrast, very few neurons demonstrated beta-galactosidase activity after injection into the adrenal gland of AdCA17lacZ and AdCA36lacZ. Therefore, 14H delta 3vhsZ is a more suitable vector than either of the adenovirus vectors tested for eliciting short-term changes in preganglionic neuron gene expression.

Identification of lamina V and VII interneurons presynaptic to adrenal sympathetic preganglionic neurons in rats using a recombinant herpes simplex virus type 1.

Although indirect evidence suggests that the control of sympathetic preganglionic neurons is mediated to a great extent through interneurons, little is known about the location, morphology or neurotransmitter phenotype of such interneurons. This limitation seriously impedes our understanding of spinal synaptic circuits crucial to control of arterial pressure and other visceral functions. We used a highly neurotropic, minimally cytopathic recombinant herpes simplex virus type-1 to study spinal "sympathetic" interneurons labelled by trans-synaptic transport of the virus from the adrenal gland in rats. Approximately 120-320 infected neurons/rat were identified by immunocytochemical detection of the viral antigen. We distinguished between virus-infected preganglionic neurons and infected interneurons by (i) their location within the spinal laminae, (ii) their size and shape and (iii) the presence or absence of immunoreactivity for the acetylcholine-synthesizing enzyme, choline acetyltransferase, a marker of sympathetic preganglionic neurons. Virus-labelled sympathetic preganglionic neurons were found within the known spinal preganglionic nuclei. Non-cholinergic, virus-labelled neurons were located throughout lamina VII and in the ventral portion of lamina V. These putative interneurons were found in the major spinal preganglionic nuclei, usually intermingled with the preganglionic neurons. Sometimes, they were located in clusters separate from the preganglionic neurons. The interneurons were approximately 15 microm in diameter, smaller than the average preganglionic neuron (diameter=25 microm), and had a few fine processes emanating from them. These non-cholinergic interneurons constituted approximately one-half of the population of virus-infected neurons. In summary, with the use of a recombinant herpes simplex virus, we identified a large number of non-cholinergic interneurons close to, or intermingled with, adrenal sympathetic preganglionic neurons. The neurotransmitter phenotype of these neurons remains to be determined but they likely integrate much of the supraspinal and primary afferent inputs to spinal preganglionic neurons that control arterial pressure and other visceral functions.