Analysis of human blood monocyte activation at the level of gene expression. Expression of alpha interferon genes during activation of human monocytes by poly IC/LC and muramyl dipeptide.

Human monocytes were activated to secrete alpha interferon (IFN-alpha) by poly IC/LC but not by other monocyte activators, such as muramyl dipeptide (MDP). In contrast, monocytes were activated to secrete fibroblast growth factor (FGF) release by MDP but not by poly IC/LC. The amount of total RNA present in unactivated and activated human monocytes was similar. Using two 32P-labeled cDNA probes (pLM001 and HuIFN-alpha 2) for human IFN-alpha genes in hybridization studies, we analyzed messenger RNA species from this gene family in activated human monocytes. After activation with poly IC/LC, two other mRNA species (2.8 and 5.5 kb) were detected in addition to the 1.0 kb mRNA normally associated with IFN-alpha secretion. Unexpectedly, monocytes activated with MDP also contained 2.8 kb IFN-alpha mRNA. There was associated with this 2.8 kb IFN-alpha mRNA, found in MDP-activated monocytes, appreciable levels of intracellular IFN-alpha activity in the absence of detectable secreted IFN-alpha. Thus the secretion of IFN-alpha in activated human monocytes can be correlated with the appearance of a 1.0 kb mRNA species after poly IC/LC exposure. Secretion appears to be defective in MDP-stimulated monocytes even though they contain active intracellular IFN-alpha apparently translated from the 2.8 kb mRNA.

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 natural history and evolution of human and simian T cell leukemia/lymphotropic viruses.

The past five years have seen significant advances in understanding the origin and evolution of human T-cell leukemia/lymphotropic virus types I and II. The highlights include the identification of human T-cell leukemia/lymphotropic virus types I and II genotypic variants in remote human populations and the discovery of widely divergent simian T-cell leukemia virus in African and Asian non-human primates.

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.

Characterization of Ca(2+)-dependent neutral protease (calpain) from human blood flukes, Schistosoma mansoni.

Calcium-dependent, neutral cysteine-proteases (calpain) were purified from human blood flukes, Schistosoma mansoni. The electrophoretic mobilities, Western blot analyses and high specificity to peptide inhibitors confirmed the presence of both calpain I and II in the purified preparation. The schistosome calpains were localized in the surface syncytial epithelium and underlying musculature. Using peptide inhibitors, calpain was shown to function as a mediator of the surface membrane synthetic process. Since there was also no immunological cross-reactivity between vertebrate and schistosome calpains using antibodies affinity-purified from native and recombinant schistosome calpains, this protease may be usefully investigated as forming the basis of a molecular vaccine against schistosomiasis.

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.

HTLV-I associated adult T cell leukemia/lymphoma: report of two cases from an Amerindian population in coastal northwest British Columbia.

Epidemiological studies of HTLV-I infection have demonstrated the presence of this virus in certain Amerindian populations in Central and South America. We have recently reported the first evidence of endemic HTLV-I infection in North American Amerindians from the coastal regions of British Columbia, Canada. While the predominant HTLV-I-associated disease observed in British Columbia Amerindians is the HTLV-I associated neurological disease (HTLV-I-associated myelopathy/tropical spastic paraparesis), we report here the first two cases of HTLV-I-associated adult T cell leukemia/lymphoma (ATL). Clinical and PCR evidence to support the diagnosis of HTLV-I-associated ATL in these two Amerindians is presented. Both cases of ATL were found in the same tribe although neither patient was directly related to each other. While reports of HTLV-I-associated ATL have been reported in Circumartic native peoples, reports of ATL in North American single ancestry Amerindians have not been previously made to our knowledge.

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.

Retroviruses and multiple sclerosis. II. Failure of gene amplification techniques to detect viral sequences unique to the disease.

Multiple sclerosis (MS) has been associated with infection by a novel human retrovirus because of its similarity to other white matter diseases caused by retroviruses and because of some serologic evidence suggesting reactivity of MS sera with antigens of HTLV-I. With the polymerase chain reaction technique, we attempted to amplify genes of HTLV-I (p24 and pol) from lymphocyte and granulocyte DNA in 44 patients with MS, 18 other neurologic or autoimmune diseases, and 11 normal controls. With primers and probes specific for these genes, we were unable to identify sequences unique to patients with MS.

Mollaret's meningitis associated with herpes simplex type 2 infection.

We describe three patients with benign recurrent aseptic meningitis (Mollaret's meningitis). For one of these cases, the episodes of meningitis were associated with herpetic outbreaks. Mollaret cells, which are a hallmark of Mollaret's meningitis, were present in the CSF from two of the three patients. In all cases, herpes simplex virus type 2 DNA was present in the CSF during the acute illness as detected by polymerase chain reaction amplification, although viral cultures from CSF were all negative. Herpesviruses, notorious for frequent and sporadic recurrence, are ideal candidates for the cause of Mollaret's meningitis.

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.

Gene transfer into sympathetic preganglionic neurons in vivo using a non-replicating thymidine kinase-deficient herpes simplex virus type 1.

The suitability of non-replicating thymidine kinase deficient herpes simplex virus type 1 expressing bacterial beta-galactosidase (tk-lacZ HSV-1) as a transfer vehicle into sympathetic preganglionic neurons in vivo was assessed. Many sympathoadrenal preganglionic neurons (451 +/- 105) with normal morphology were identified using beta-galactosidase histochemistry two days after inoculation of tk-lacZ HSV-1 into the adrenal gland of hamsters. Beta-galactosidase activity co-localized with nicotinamide adenine dinucleotide phosphate-diaphorase-positive sympathetic preganglionic neurons in the nucleus intermediolateralus, pars principalis. The maximal number of beta-galactosidase expressing neurons was found two days post-inoculation but this number dropped dramatically after this time. An inflammatory infiltrate was abundant around infected neurons and in the white matter at five days and infected neurons appeared morphologically abnormal. At 26 days, the infiltrate was still present but no infected sympathoadrenal preganglionic neurons were detected. Approximately 25% fewer nicotinamide adenine dinucleotide phosphate-diaphorase-positive neurons in the nucleus intermediolateralis, pars principalis were counted ipsilaterally than contralaterally in animals infected for 14, 21 or 26 days with tk-lacZ HSV-1, compared to the 3% difference in animals mock-infected for 26 days. Approximately 33% of the estimated number of sympathoadrenal preganglionic neurons infected with tk-lacZ HSV-1 at five days were apoptotic or necrotic. About 60% of neurons infected with tk-lacZ HSV-1 at two days no longer expressed nicotinamide adenine dinucleotide phosphate-diaphorase at 14-26 days. In conclusion, the non-replicating thymidine kinase deficient HSV-1 was efficiently retrogradely transported from the adrenal gland to infect sympathoadrenal preganglionic neurons. These gene transfer experiments using tk-lacZ HSV-1 suggest that foreign gene expression in sympathetic preganglionic neurons in vivo may be maximal two days after inoculation when beta-galactosidase was expressed in the greatest number of sympathetic preganglionic neurons. After two days, fewer neurons expressed beta-galactosidase and the presence of tk-lacZ HSV-1 appeared to be altering protein expression in sympathetic preganglionic neurons and/or leading to the demise of the infected neuron.

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.

Identification of spinal interneurons antecedent to adrenal sympathetic preganglionic neurons using trans-synaptic transport of herpes simplex virus type 1.

Control of sympathetic preganglionic neurons appears to be mediated, in part, through polysynaptic pathways using spinal interneurons. To identify spinal interneurons antecedent to adrenal sympathetic preganglionic neurons, we injected herpes simplex virus type 1 into the adrenal gland of hamsters as this virus is an effective trans-synaptic tracer of neural pathways. After a three day survival period, immunocytochemistry was used to visualize virus-infected spinal cord cells. Infected sympathetic preganglionic neurons with somata that were either kite-shaped, elliptical or fusiform and that had extensive dendrite arbors were identified as well as a group of smaller round cells with finer processes. For comparison, in additional hamsters, labelling with the retrograde tracer Fluoro-Gold and histochemical reactions for the enzyme nicotinamide adenine dinucleotide phosphate-diaphorase were used to identify sympathetic preganglionic neurons. Sympathetic preganglionic neurons identified with Fluoro-Gold or herpes virus were present mostly in the nucleus intermediolateralis, pars intermediolateralis and nucleus intermediolateralis, pars funicularis of the spinal cord. The smaller herpes virus-infected cells were found mostly medial to the preganglionic neurons in lamina VII and also dorsally in lamina V of the spinal cord. Assessing immunoreactivity for glial fibrillary acidic protein demonstrated that the smaller herpes virus-infected cells were not reactive astrocytes. Furthermore, these cells were immunoreactive for two neuronal markers, neuron-specific enolase and for microtubule-associated protein 2. These findings suggest that these smaller round cells with finer processes are distinct from sympathetic preganglionic neurons and astrocytes and may be interneurons antecedent to the sympathetic preganglionic neurons.

Identification of renal sympathetic preganglionic neurons in hamsters using transsynaptic transport of herpes simplex type 1 virus.

Herpes viruses have been used as retrograde transsynaptic tracers to identify pathways from the CNS to specific target tissues. We used herpes simplex virus to identify central nervous system neurons responsible for control of the kidney. Herpes simplex type 1 or herpes simplex type 2 was injected into rat kidneys and herpes simplex type 1 was microinjected into hamster and guinea pig kidneys. After three to seven days, ganglia, spinal cords and brains were examined using immunohistochemistry to visualize the virus-infected neurons. Our first experiments demonstrated that rats were not susceptible to infection with neurotropic strains of herpes simplex type 1. Injections of a wildtype strain of herpes simplex type 2 into rat kidneys led to nonspecific infection of many central nervous system neurons and glia. In contrast, herpes simplex type 1 injections in hamsters and guinea pigs caused specific infection of limited numbers of neurons in approximately one-third of the animals and the study was continued using hamsters. Sympathetic preganglionic neuron labelling was found in the ipsilateral intermediolateral cell column of the spinal cord as well as the lateral funiculus. Most infected preganglionic neurons were located in the seventh to the ninth thoracic spinal segments. Infected neurons were not found in the dorsal or ventral horn of the spinal gray matter and only one or two cells were found in the brainstem. Sympathetic preganglionic neuron morphology was usually normal, showing detailed dendritic arborizations, and lysis was infrequent. Small infected cells were sometimes observed close to sympathetic preganglionic neurons. Because herpes simplex type 1 virus was not detected immunocytochemically in ganglionic neurons in these same hamsters, the polymerase chain reaction was used in some additional hamsters to detect viral DNA in the T12 and T13 chain ganglia and splanchnic ganglia ipsilateral to the kidney injected with herpes simplex type 1. Finally, the overall distribution of renal postganglionic and splanchnic preganglionic neurons in hamsters was examined for comparison to the number and locations of virus-labelled neurons. Retrograde transport of the fluorescent dye FluoroGold demonstrated that (i) renal postganglionic neurons are distributed in the T10-L1 chain ganglia and in the prevertebral splanchnic ganglion and (ii) splanchnic preganglionic neurons are located in the T3-T12 spinal segments, predominantly in the intermediolateral and funicular spinal autonomic nuclei. In conclusion, herpes simplex type 1 virus infected an exclusive population of "renal" neurons in hamsters without lysis and with little cellular reaction to the infection after a survival period of three days, permitting these neurons to be studied in detail.

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.

Delivery of a foreign gene to sympathetic preganglionic neurons using recombinant herpes simplex virus.

Two recombinant herpes simplex type 1 viruses expressing beta-galactosidase (encoded by the Escherichia coli lacZ gene) inserted into the unique long 41 (encoding virus host shutoff) or unique short 5 (encoding glycoprotein J) open reading frames were generated. Purified recombinants or wild-type herpes simplex type 1 were injected into the left adrenal gland of hamsters. Three days later, virus-infected neurons were detected in spinal cord sections from all infected hamsters. Neurons were visualized with beta-galactosidase histochemistry in spinal cord sections from hamsters infected with either of the recombinants but not with the wild-type virus. Wild-type virus could only be detected with immunocytochemistry. Insertional mutagenesis into the unique long 41 or unique short 5 regions of the herpes simplex genome by lacZ did not disrupt the neurotropic properties of the virus. Both recombinant viruses labelled the central nervous system sympathoadrenal preganglionic neurons as well as brainstem neurons. Because the virus host shutoff recombinant more readily crossed synapses to reach the brainstem compared to the glycoprotein J recombinant, the presence of glycoprotein J may facilitate cell to cell transmission in vivo. Both recombinants may be useful for the study of synaptic organization of neural circuits. Our recombinant viruses were less lytic yet neurovirulent after mutation of either glycoprotein J or virus host shutoff of herpes simplex virus type 1 wild-type. These recombinant viruses express the bacterial beta-galactosidase which is readily detectable using simple histochemistry. Inoculation of the adrenal gland or kidney with these viruses led to clear labelling of spinal cord cells. These viruses may be useful markers of specific neural circuits.

Simultaneous identification of two populations of sympathetic preganglionic neurons using recombinant herpes simplex virus type 1 expressing different reporter genes.

We generated neurotropic herpes simplex type 1 viruses expressing human placental alkaline phosphatase and studied the utility of this enzyme as a marker of infected neurons. The neurotropism of these viruses was assessed by their ability to infect sympathetic preganglionic neurons after adrenal injection in hamsters. The transneuronal transfer of these viruses was examined by their ability to cross the peripheral synapse from the kidney to renal preganglionic neurons or to cross the central synapse from the adrenal gland to the medulla oblongata. Finally, we injected an alkaline phosphatase-expressing herpes simplex virus into the adrenal gland and a beta-galactosidase-expressing herpes simplex virus (US5gal) into the muscular wall of the small intestine to label two neural circuits in one animal and to assess the feasibility of a dual-virus labelling system. The alkaline phosphatase gene was inserted into the glycoprotein J locus or the virus-induced host shut-off locus in the herpes simplex genome to create viruses which replicate (gJHAP HSV or vhsHAP HSV) or into the thymidine kinase locus to generate a virus that does not replicate in neurons in vivo (TK- HAP HSV). Each of the three viruses was retrogradely transported from the adrenal gland of hamsters to sympathetic preganglionic neurons, suggesting that the neurotropism of these viruses was maintained. gJHAP HSV travelled transneuronally from the kidney to sympathorenal preganglionic neurons and from the adrenal gland to neurons in the rostral ventrolateral medulla. Neuronal infection with alkaline phosphatase-expressing virus could be identified using histochemistry but detailed morphology of these neurons was not revealed. However, staining by anti-herpes simplex virus immunoperoxidase demonstrated that they had normal morphology. Identification of two distinct neural circuits in one animal was achieved with our dual-virus labelling system. The nonreplicating TK- HAP HSV was used in combination with US5gal to identify intestinal and adrenal sympathetic preganglionic neurons. The beta-galactosidase-expressing intestinal neurons were labelled bilaterally in the nucleus intermediolateralis, pars principalis, and alkaline phosphatase-expressing adrenal neurons were found ipsilaterally. Some clusters of sympathetic preganglionic neurons in the nucleus intermediolateralis, pars principalis contained mostly intestinal sympathetic preganglionic neurons and a few adrenal sympathetic preganglionic neurons. In other areas, the opposite pattern occurred. About 3-7% of the labelled sympathetic preganglionic neurons were double-labelled by both markers. The distinct and crisp morphology and dendritic processes of neurons stained by beta-galactosidase histochemistry contrasted with the partial staining of neurons by alkaline phosphatase, revealing beta-galactosidase as a better marker of infected neurons. In conclusion, alkaline phosphatase-expressing herpes simplex viruses are yet neurotropic after insertion of this marker enzyme into any of three different loci of the herpes simplex genome. One replicating alkaline phosphatase-expressing virus travelled transneuronally. These alkaline phosphatase-expressing herpes simplex virus can be used together with beta-galactosidase-expressing herpes simplex viruses to determine the target specificity of sympathetic preganglionic neurons controlling visceral organs or can be used to express two different recombinant genes in two targeted neuronal populations. This study suggests that sympathetic preganglionic neurons controlling the intestine and adrenal gland are almost completely distinct.

Nucleotide sequence analysis of an HTLV-I isolate from a Chilean patient with HAM/TSP.

Isolates of HTLV-I have been characterized from a number of different regions of the world; however, there has not been a nucleotide sequence analysis of an HTLV-I isolate from a South American country. Reported here is an individual from Chile identified with the HTLV-I-associated neurological disease HAM/TSP. The sera and the nucleic acid sequence of the HTLV-I present in peripheral blood lymphocytes from this Chilean HAM/TSP patient over a two year period are characterized. During this time, the patient's condition grew progressively worse. While the serological profile of this patient was unremarkable in comparison with other HAM/TSP patients previously described, nucleic acid sequence analysis identified two nucleotide positions which contained nucleotides unique to this Chilean isolate. The nucleotide sequence analysis also indicates that the Chilean HTLV-I isolate is more closely related to Caribbean and Japanese isolates of HTLV-I than to the African and U.S. isolates described so far.

Production of immunogenic HIV-1 viruslike particles in stably engineered monkey cell lines.

A proviral fragment from human immunodeficiency virus type 1 (HIV-1) (LAV-1BRU) containing only protein-coding information, was expressed in COS cells using constitutive promoters in transient and stable transfection experiments. The presence of viruslike particles in cell supernatants was verified by Western blot analysis, density gradient centrifugation, and electron microscopy. Transfection of Vero cells with a similar construct employing the human metallothionein promoter led to the isolation of stable cell lines exhibiting inducible viruslike particle expression in response to cadmium chloride treatment. Induction ratios for viruslike particle expression were in excess of 1000-fold with production levels of p24 core antigen as high as 0.6 mg/L per 24 h. HIV-1 viruslike particles were immunogenic in mice, leading to strong envelope and core-specific humoral responses after two immunizations. The development of stable cell lines expressing significant quantities of HIV-1 viruslike particles offers an alternative to the use of live virus vectors for the production and evaluation of particle-based AIDS vaccines.