Cyclosporin A reduces the in¯ammatory response to amulti-mutant herpes simplex virus type-1 leading toimproved transgene expression in sympatheticpreganglionic neurons in hamsters

P Joy Mabon1,3, Lynne C Weaver1,3 and Gregory A Dekaban*,2,4

1Neurodegeneration Research Group, The John P. Robarts Research Institute, 100 Perth Drive, PO Box 5015, London,Ontario, Canada N6A 5K8; 2Gene Therapy and Molecular Virology Group, The John P. Robarts Research Institute, 100Perth Drive, PO Box 5015, London, Ontario, Canada N6A 5K8; 3Department of Physiology, The University of WesternOntario, London, Ontario, Canada and 4Department of Microbiology and Immunology, The University of WesternOntario, London, Ontario, Canada

Herpes simplex virus type 1 (HSV-1) based vectors hold great promise for genetransfer to CNS neurons. Problems such as loss of transgene expression, vector-associated cytotoxicity and the immune response to the vector or encodedtransgene still remain obstacles to success. We used a replication-defective,HSV-1 vector (14HD3vhsZ) that was engineered to have reduced cytotoxicityand express recombinant b-galactosidase. A previous study in our laboratoryshowed no evidence for cytotoxicity in infected neurons although anin¯ammatory in®ltrate occurred around infected cells and transgene expres-sion was lost between 5 and 8 days. The immune response consisted of aprimary response at the site of inoculation (adrenal gland), and a secondaryimmune response in the spinal cord around infected adrenal sympatheticpreganglionic neurons due to retrograde transport of the vector. We testedwhether conventional immunosuppressants could reduce the secondaryimmune response, leading to improved transgene expression at the secondaryCNS site. 14HD3vhsZ was injected into the adrenal gland in hamsters 1 day afterimmunosuppressant treatment began. Non-drug treated, 14HD3vhzZ-infectedhamsters were used as controls. Cyclosporin A administration led to the mostpersistent b-galactosidase activity in neurons at 5 and 8 days. Methylpredni-solone treatment resulted in the greatest reduction in the in¯ammatory cellin®ltrate but the numbers of infected neurons did not increase concomitantly.This suggested no direct relationship between extent of the in¯ammatory cellin®ltrate and level of transgene expression. These data demonstrate thepotential of cyclosporin A as an immunosuppressant adjunct treatment forHSV-1 vector-mediated gene transfer from a peripheral site to neurons in thespinal cord.

Keywords: cyclosporin A; in¯ammation; methylprednisolone; recombinantherpes simplex virus type 1; spinal cord; transgene expression

Introduction

Recombinant herpes simplex type 1 viruses (rHSV-1) have elicited considerable interest for theirpotential as a gene transfer vector to neurons ofthe central nervous system (CNS). HSV-1 has manyfeatures well suited for this task. The genetic

organisation of this large human virus and theproperties of replication are well characterised.Large portions of the HSV genome (up to 30 kb)can be replaced with genes of interest (Huard et al,1997). HSV-1 is naturally neurotropic, able to infecta wide range of mitotic and postmitotic cells, andmaintain a lifelong latent or `quiescent' infection insensory neurons (Fink et al, 1996; Glorioso et al,1995a, b; Kennedy and Steiner, 1993). Moreover,HSV and replication defective rHSV, with the

*Correspondence: GA DekabanReceived 7 July 1998; revised 18 September 1998; accepted 12October 1998

Journal of NeuroVirology (1999) 5, 268 ± 279ã

http://www.jneurovirology.com

1999 Journal of NeuroVirology, Inc.

appropriate helper cell lines, can generate the hightiter viral stocks (4108 p.f.u.) necessary to deliver agene ef®ciently and speci®cally to a large number oftarget neurons (Fink et al, 1996; Glorioso et al,1995a, b). Herpes viruses have been used success-fully by several investigators to express potentiallytherapeutic or reporter genes in the mammalianbrain (Bloom et al, 1995; Wolfe et al, 1996; Gloriosoet al, 1994) and spinal cord (Hong et al, 1995;LeVatte et al, 1997b; Mabon et al, 1997). The goal ofHSV-1 vector systems is to permit short or longterm regulated gene expression at therapeutic levelsin the absence of immune or virus-mediatedcytotoxicity. Presently, however, these criteria havenot been satis®ed fully. The immunological con-straints, in particular, remain a signi®cant obstacleto success.

Sympathetic preganglionic neurons (SPNs) arethe last site for integration of synaptic inputs thatregulate systemic arterial pressure. Ultimately, genetransfer to SPNs may allow better control of bloodpressure after spinal cord injury, for example, andstrategies to promote longer expression of poten-tially therapeutic genes by rHSV-1 vectors in SPNsare important. SPNs are not easily accessible byintraspinal injection since they are organised in anarrow column along the length of the thoracolum-bar spinal cord. Axons of SPNs converge inperipheral organs and ganglia that can be used assites for vector inoculation. Peripheral inoculationof the vector at a primary site for retrogradeneuronal transport is necessary for selective target-ing of these neurons (secondary site; Figure 1).

To utilise HSV vectors for gene delivery to theCNS, both direct and indirect virus-induced cyto-toxic effects must be addressed. In this study weused the 14HD3vhsZ rHSV-1 vector (Johnson et al,1994) to infect adrenal SPNs. This vector wasengineered to produce less cytotoxicity than the®rst generation replication-defective recombinantherpes simplex viruses (thymidine kinase de®cientrHSV-1 (LeVatte et al, 1997a)) while maintainingstrong neurotropism. Deletion or inactivation ofthree of the major genes responsible for cellularcytotoxicity, rendered 14HD3vhsZ replication-de®-cient in neurons. LeVatte et al (1998) demonstrateda lack of cytotoxicity of 14HD3vhsZ in SPNs in vivoand minimal toxicity in differentiated PC12 cells invitro. Indeed, in vivo there was no loss of SPNs oralteration in the levels of the important cellularenzymes, choline acetyltransferase or nicotinamideadenine dinucleotide phosphate-diaphorase activ-ity at 14 days after infection with 14HD3vhsZ. Thegreatest number of b-galactosidase (b-gal) expres-sing SPNs (approximately 300) in the thoracicspinal cord occurred at 3 days post inoculationwith 14HD3vhsZ into the adrenal gland (LeVatte etal, 1998). This represents a large percentage of thetotal population of adrenal SPNs since studies usingconventional tracers showed a range of 300 ± 1000

neurons on a single side of the cord (Schramm et al,1975; Pyner and Coote, 1994). Further improve-ments are still required, though, since an in¯am-matory in®ltrate appeared around infected SPNs by5 days and expression of the marker gene lacZ wasessentially lost by 8 days post-infection with14HD3vhsZ (LeVatte et al, 1998). Immune re-sponses to the vector or transgene product may beresponsible for driving the vector into latency(Wood et al, 1994). Thus, strategies of immunomo-dulation must be developed to promote longerexpression of virally transferred genes.

The characteristics of in¯ammation in the CNSand ganglia due to peripheral infection with HSV-1have been outlined in several reports (Liu et al,1996; Wood et al, 1994). Both wildtype HSV-1 andrecombinant HSV-1 vectors evoke similar immuneresponses consisting of a primary response at thesite of inoculation (i.e. adrenal gland) and asecondary immune response in the spinal corddue to retrograde transport of the vector. Brie¯y,this secondary response in the spinal cord ischaracterised by perivascular in®ltration of macro-phages, neutrophils, T lymphocytes (both CD4+ andCD8+), induction of MHC class I and II moleculesand production of interferon gamma (IFNg) andtumour necrosis factor-alpha (TNF-a) (Wood et al,1994). A similar response pattern is also observed inthe animal model of multiple sclerosis, experimen-tal allergic encephalitis (EAE). Injection of ence-

Figure 1 Schematic diagram of the thoracic spinal cord(secondary site) and innervation of the adrenal gland (primarysite) by sympathetic preganglionic neurons.

CsA reduces HSV-induced in¯ammation in spinal cordPJ Mabon et al

269

phaletogenic antigen at a peripheral or primary siteleads to perivascular in¯ammation of the CNS(secondary site; Zamvil and Steinman, 1990). InHSV-1 infection of ganglia and in EAE, TNF-a andmacrophages appear to be major participants in theimmune response at the secondary site (Johnson etal, 1994; Liu et al, 1996; Martin et al, 1995).

In addition to its proven bene®t in organtransplantation (Perico and Remuzzi, 1997), Cyclo-sporin A (CsA) is a potent immunosuppressivecompound that has been shown to be protective inthe chronic EAE model (Calder et al, 1987). CsA isable to prevent EAE and the concomitant in¯am-matory response in the CNS, if it is administered ator before immunisation with encephaletogenicantigen and adjuvant, despite its inability to crossthe blood brain barrier (Branisteanu et al, 1995;Huitinga et al, 1995; Mustafa et al, 1993). Two otheref®cacious immunosuppressants, methylpredniso-lone (MP) and azathioprine (AZA) are also widelyused in clinical settings and animal experiments fortheir ability to suppress globally the immuneresponse of the host. In addition, these drugs havebeen shown to ameliorate EAE (Bolton, 1995).

These conventional immunosuppressants haveprevented the induction of EAE or reduced theseverity of the immune response in the CNS(secondary site) in the chronic EAE model. Wehypothesized that they also may prevent orreduce the vector or transgene-associated immuneresponse in our model of retrograde transport of14HD3vhsZ from the adrenal gland (primary site)to the spinal cord (secondary site). We assessedthe level of gene expression by counting numbersof SPNs expressing the marker gene product, b-gal, at 5 and 8 days. We assessed extent ofin¯ammation by calculating areas of in¯ammatoryin®ltrate around SPNs and areas of perivascularcuf®ng in drug-treated and non drug-treatedanimals.

Results

Effect of immunosuppressants on the morphologyand number of SPNs exhibiting b-gal activity at 5days post-infection with 14HD3vhsZIn the absence of immunosuppressant treatment, anaverage of 45+9 SPNs expressing b-gal wasobserved throughout the entire length of thethoracic spinal cord in four hamsters (Figure 2).The majority of these SPNs showed incomplete®lling of neuronal cell bodies with reaction productand few visible dendrites as revealed by X-galhistochemistry (Figure 3A and B), although someSPNs exhibited b-gal reaction product entirely®lling kite-shaped somata and elaborating severalmajor dendrites. CsA treatment led to a signi®cantimprovement in the persistence of b-gal expressionin SPNs averaging 126+23 in the thoracic region of

the cord in six hamsters (Figure 2). Moreover, inCsA treated animals, robust b-gal activity ininfected SPNs revealed normal kite-shaped somataand exhibited many elaborate dendritic processes(Figure 3C and D). No appreciable difference inquality of transgene expression (represented bynumber and morphology of SPNs expressing b-gal)was evident at a lower dose (1 mg/kg/day) of MPtested in three animals compared to control values.Immunosuppression by MP at a higher dose (3 mg/kg/day, i.p.) tended to increase the numbers of SPNsexpressing b-gal (95+30 SPNs; Figure 2) in sixhamsters, although these values were not signi®-cantly different than control animals. Expression ofthe marker enzyme in neurons of MP treatedhamsters revealed the morphology of the neuronsomata and extensive dendritic arbors in greatdetail (Figure 3E and F). Daily injections of AZA(10 mg/kg/day; i.p.) did not increase the number ofSPNs (30+11 SPNs) expressing the transgene infour hamsters compared to control animals (Figure2). Some SPNs expressing b-gal in AZA treatedhamsters exhibited kite-shaped somata and bluereaction product in dendritic processes, but manySPNs exhibited less b-gal activity in smaller areas ofthe cell body and fewer dendritic processes than inCsA or MP treated hamsters (data not shown). Inaddition, in later studies we increased the dose ofAZA to 20 mg/kg/day, i.p., but still found noimprovements in any of the measured parameters(data not shown). Daily immunosuppression by acombination therapy with a lower dose of CsA(35 mg/kg; i.m.) and the higher dose of MP (3 mg/kg; i.p.) led to no signi®cant improvement in thenumber of SPNs exhibiting b-gal activity (62+3

Figure 2 Mean numbers of SPNs visualised by X-gal histo-chemistry for b-galactosidase activity in the intermediolateralcell column (IML) of spinal cord segments T1-13 at 5 days post-infection with 14HD3vhsZ. From left to right, bars representnon-drug treated control hamsters (n=4), CsA treated (50 mg/kg)(n=6), MP 1 mg/kg (n=3), MP 3 mg/kg (n=6), AZA 10 mg/kgtreated hamsters (n=4) and CsA/MP 35/3 mg/kg (n=3). Valuesrepresent the mean+standard error of the mean (s.e.m.). The *indicates a signi®cant difference (P50.05) compared to non-drugtreated control animals.

CsA reduces HSV-induced in¯ammation in spinal cordPJ Mabon et al

270

SPNs; Figure 2) in three hamsters compared tocontrol animals.

Effect of immunosuppressants on the immuneresponse at the secondary site at 5 days post-infection with 14HD3vhsZIn non-immunosuppressed control hamsters, arobust in¯ammatory in®ltrate occurred aroundinfected SPNs (Figure 3A and B) with a mean areaof in¯ammation per neuron of 0.024+0.002 mm2

(Figure 4A). Perivascular cuf®ng was evident on theinfected side of the spinal cord (Figure 3A, whitearrows) with a mean area of 6.12+1.28 mm2 (Figure4B). Treatment with CsA reduced signi®cantly thein¯ammatory in®ltrate surrounding infected SPNsto 0.009+0.002 mm2 (Figures 3C, D and 4A) andtended to reduce the area of perivascular cuf®ngcompared to control hamsters (4.17+0.88 mm2;Figure 3C and D arrows, Figure 4B). The lower doseof MP (1 mg/kg/day) did not alter the area ofin¯ammation around infected SPNs (0.021+0.002mm2) compared to control values. However, the areaof perivascular cuf®ng of immune cells wassigni®cantly reduced to 2.79+1.16 mm2 (Figure4B). Treatment with MP (3 mg/kg/day) signi®cantlyreduced the average area of in¯ammation around

infected SPNs to 0.009+0.003 mm2 (Figure 4A).Moreover, the area of perivascular cuf®ng also wassigni®cantly reduced to 1.06+0.36 mm2 (Figure4B). Treatment with AZA (10 mg/kg) however, didnot reduce in¯ammation around infected SPNs(0.032+0.009 mm2; Figure 4A). AZA reduced thearea of perivascular cuf®ng to 2.940+0.848 mm2

(Figure 4B) compared to non-immunosuppressedhamsters. Combination drug therapy with CsA andMP (35 mg/kg; 3 mg/kg) resulted in no signi®cantreduction in the in¯ammation surrounding infectedSPNs (0.071+0.003 mm2; Figure 4A), or area ofperivascular cuf®ng (4.19+0.80 mm2; Figure 4B)compared to non-immunosuppressed control va-lues.

Effect of immunosuppressants on the morphologyand number of SPNs exhibiting b-gal activity at 8days post infection with 14HD3vhsZDaily administration of CsA (50 mg/kg; i.m.) in-creased signi®cantly the average number of SPNsexpressing b-gal to 19+7 at 8 days post-inoculationwith 14HD3vhsZ in four hamsters compared to noexpression at this time in our previous study (LeVatteet al, 1998). SPNs in the thoracic spinal cord stillexhibited a robust reaction product elaborating the

Figure 3 Photomicrographs of infected SPNs in horizontal sections of thoracic spinal cord 5 days post-infection with 14HD3vhsZ.Non-drug treated control animals (A,B) exhibit b-gal reaction product incompletely ®lling the cell bodies and dendrites. An extensivein¯ammatory in®ltrate and perivascular cuf®ng (white arrows) are present. CsA-treated (C,D) and MP-treated (E,F) animals exhibitnormal cellular morphology with elaborate dendritic processes extending from kite-shaped somata. There is indication of anin¯ammatory in®ltrate around infected SPNs and perivascular cuf®ng (white arrows) on the infected side of the cord but it is markedlyreduced compared to control animals. Calibration bar in A is 50 mm and pertains to A, C and E. Calibration bar in B is 20 mm andpertains to B, D and F.

CsA reduces HSV-induced in¯ammation in spinal cordPJ Mabon et al

271

neuronal somata and dendritic process (Figure 5Aand B). Daily administration of MP (3 mg/kg; i.p.)resulted in 9+2 SPNs exhibiting b-gal activityfollowing adrenal injection of the 14HD3vhsZvector in six hamsters.

Effect of immunosuppressants on the immuneresponse at the secondary site at 8 days post-infection with 14HD3vhsZIn non-immunosuppressed control hamsters, wecould not calculate the area of in¯ammatoryin®ltrate surrounding infected SPNs since nonewere evident at 8 days post-infection with therHSV-1 vector. Daily injection of CsA led to anaverage area of in¯ammation per neuron of0.007+0.003 mm2. Treatment with MP (3 mg/kg/day) resulted in a mean area of in®ltrates aroundinfected SPNs of 0.013+0.003 mm2. Perivascular

cuf®ng was evident on the side of the cordipsilateral to injection with 14HD3vhsZ with meanarea of 0.92+0.21 mm2 in non-immunosuppressedhamsters. Treatment with CsA or MP did notsigni®cantly reduce the area of perivascular cuf®ngat this time point compared to control values. Meanareas were 2.05+1.05 mm2 and 0.79+ 0.09 mm2

respectively.

Discussion

Viral vectors have great potential for transfer oftherapeutic genes to various tissues to amelioratecertain neurologic conditions. Despite good initialsuccess with high infection ef®ciencies and highinitial expression levels, many groups have reporteda quick decline in vector-mediated transgeneexpression in targeted tissues (Yang et al, 1994;Herz and Gerard, 1993; Keir et al, 1995). At thesame time, experimental studies have demonstratedthe best success with either newborn or immuno-de®cient animals (Davidson et al, 1993; Hermensand Verhaagen, 1997). Since these animals lack amature or properly functioning immune system, theimmune response could be at least partly respon-sible for loss of transgene expression by the vector.Clinically useful gene transfer protocols for treat-ment of various neurological disorders will requirestable and prolonged expression of therapeuticgenes. Other studies have reported success inimproving the length of vector-mediated markergene expression with various immunosuppressants.Kaplan and Smith (1997) reported, for example,improvement in the longevity of transgene expres-sion and ability to readminister adenoviral vector inmouse lung, resulting from transient immunosup-pression with deoxyspergualin (Guerette et al,1995). Similarly, Durham et al (1997) used FK506to prolong transgene expression in the brainfollowing adenovirus-mediated gene transfer. How-ever, these studies were carried out using variousadenovirus vectors to deliver transgenes to organsor were injected directly into the brain. In certainconditions, speci®c populations of spinal neuronsmay require the transfer of therapeutic genes in theabsence of invasive techniques such as stereotacticinjection that results in injury and a concomitantin¯ammatory response at the injection site. Genetransfer to speci®c populations of spinal cordneurons would be attained effectively via HSVvectors that naturally undergo retrograde transportfrom a de®ned peripheral inoculation site. Thus,HSV vectors can be targeted to speci®c sites in thespinal cord and other CNS areas without direct CNSinjection. While stereotactic injection and the use ofadeno, adeno-associated vectors or lentiviral vectorswith neuron-speci®c promoters will allow a certaindegree of targeting of neuronal populations, thebiology of these viruses does not normally include

Figure 4 (A) Histograms depict mean areas of in¯ammation perSPN infected with 14HD3vhsZ at 5 days in the ®ve treatmentgroups (format as in Figure 2). An MCID system was used totrace the extent of in¯ammation around SPNs and the area ofin¯ammation was calculated per animal. This value wasconverted to area of in¯ammation per SPN by dividing thisarea by the total number of SPNs labelled by X-gal histochem-istry for that animal. (B) depicts mean areas of perivascularcuf®ng (PC) on the infected side of the spinal cord. Areas of PCwere traced using the MCID system and summed per animal.Mean areas were calculated per treatment group and comparedto non-drug treated control values. Numbers are means+stan-dard error. The * indicates a signi®cant difference (P50.05)compared to non-drug treated control animals.

CsA reduces HSV-induced in¯ammation in spinal cordPJ Mabon et al

272

ef®cient retrograde transfer from a peripheral site toneurons in the CNS. For example, various adeno-virus vectors have been used for gene transfer to theCNS by direct injection (Davidson and Bohn, 1997;Peltekian et al, 1997; Hermens and Verhaagen,1997). Previous work in our laboratory, however,reported that adenoviruses were not ef®cient inretrograde transport from the adrenal gland to SPNsin the spinal cord (LeVatte et al, 1998). HSV-1vectors, then, may prove to be more useful for genedelivery to speci®c sets of neurons from a de®nedperipheral site outside the CNS and thus animmunomodulation system designed to prolongtransgene expression in this model will be im-portant.

This study is, to our knowledge, the ®rst to reportthe use of immunosuppression to prolong transgeneexpression from a rHSV-1 vector at a secondary sitein the CNS following injection at a peripheral siteoutside the CNS. In our study, the potential ofconventional immunosuppressants was examinedfor their ability to prolong transgene expression bythe 14HD3vhsZ vector in SPNs. A clear advantage ofimmunosuppression by CsA was found as twice asmany SPNs expressed b-gal at 5 days post-infectionwith 14HD3vhsZ than were found in non-immuno-suppressed animals. Thus, it was possible at 5 dayspost-infection to have recombinant gene expressionin 100 ± 150 SPNs out of a possible 300 ± 1000adrenal SPNs on one side of the spinal cord(Schramm et al, 1975; Pyner and Coote, 1994).Further, daily CsA increased the length of time b-galpositive SPNs could be detected to 8 days, the timewhen b-gal activity had been lost in this system in aprevious study (LeVatte et al, 1998).

The mode of action of CsA is well known since itis the most widely used drug in organ transplanta-tion and neural transplant studies (Borlongan et al,1996). It selectively reduces the synthesis of IL-2 byactivated helper T-cells (Th T-cells) and thusinhibits activation of resting T-lymphocytes (Lem-strom et al, 1996). Macrophages, as antigen pre-senting cells, have been shown to play a critical rolein this process (Benson and Ziegler, 1989; Palay etal, 1986). In addition to blocking the proliferativeresponse of T-cells and induction of IL-2 synthesis,it inhibits the ability of cytotoxic T-cells to respondto IL-2. Moreover, CsA has been shown to abrogateboth the recruitment and activation of macrophages(Berkowitz-Balshayi et al, 1995). Other groups havereported the involvement of CsA in the regulation ofT-cell-epithelial cell adhesion by altering leuko-cyte-function associated antigen-1 (LFA-1) and itscounter-receptor intercellular adhesion molecule-1(ICAM-1) expression (Frishberg et al, 1996). Due toCsA's known inhibition of resting T-lymphocyteactivation (Lemstrom et al, 1996), our resultssuggest that an important component of thevector-induced and/or transgene-induced immuneresponse is cell-mediated and likely involves T-cells and macrophages. CsA's suppressive effect onthe macrophage's ability to present antigen is likelythe critical event at both the primary and secondarysites of HSV vector infection in our model.Normally, as described for other models of virusinfection (Kasaian and Biron, 1990), macrophage-mediated antigen presentation leads to Th T-celland cytotoxic T-cell activation and IL-2 mediatedproliferation at 3 ± 5 days post-infection. Thisresponse peaks at 7 days and diminishes by 9 days.

Figure 5 Photomicrographs of an SPN in the IML of a horizontal section of thoracic spinal cord taken from a hamster at 8 days post-infection with 14HD3vhsZ and receiving daily injections of CsA (50 mg/kg, i.m.). White arrow indicates residual perivascular cuf®ng.Calibration bar in A is 50 mm, calibration bar in B is 20 mm.

CsA reduces HSV-induced in¯ammation in spinal cordPJ Mabon et al

273

This time course closely re¯ects the time course ofthe vector and/or transgene induced in¯ammatoryresponse in the spinal cord in the absence ofimmunosuppression based on the results presentedpreviously and in this report (LeVatte et al, 1997a,1998). The early antiviral macrophage and T-cellresponse triggered in immunocompetent hamstersand blocked by CsA could indeed be limiting theduration of vector-encoded gene expression. Theresidual in¯ammatory response that we observedaround infected SPNs could be due to the arrival ofactivated NK cells and neutrophils which usuallyarrive before activated T-cells at 3 days post-infection (Kasaian and Biron, 1990). CsA does notblock the activation of NK cells (Kasaian and Biron,1990). As mentioned previously, a study involvingan adenoviral vector found that adenoviral vector-mediated gene expression was prolonged followingdirect injection into the CNS in T-cell de®cientnude rats compared to immunocompetent Wistarrats (Hermens and Verhaagen, 1997), furtheremphasising the importance of cell-mediated im-munity on expression of vector-encoded genes.

Methylprednisolone signi®cantly reduced in-¯ammation around infected SPNs and the areaof perivascular cuf®ng, an effect which tended tocoincide with more pronounced expression of b-gal at 5 days. Corticosteroids, such as MP, inhibitseveral aspects of T-cell immunity mostly viainhibition of cytokine expression at the transcrip-tional and posttranscriptional level (Almawi et al,1996; Chaudhary and Avioli, 1996). They also aretoxic to lymphocytes when given systemicallyand they inhibit the in¯ammatory activity ofmacrophages and other phagocytic cells, furtherhindering the immune response to pathogens(Almawi et al, 1996). In addition, steroids reducethe ability of macrophages to respond to lympho-cyte-derived signals (Lemstrom et al, 1996). Thesuccess of MP in enhancing transgene expressionmay have resulted from a reduction in numberand action of macrophages, which have beendemonstrated to suppress HSV gene expression(Wu et al, 1993).

Daily administration of AZA at both doses testeddid not improve transgene expression or decreasethe in¯ammatory response around infected SPNs.Azathioprine is a powerful anti-metabolite thatinhibits the development of both humoral andcellular immunity by interfering with the prolifera-tion of activated lymphocytes (Lemstrom et al,1996). Reports indicate that the bioavailability ofAZA can be low and very mutable with highlyvariable time to peak, maximum concentration,clearance and half-life (Daar, 1995). Moreover, lowpenetration into the CNS and/or lack of conversionto its active metabolite in tissue might explain whythe immune response is seemingly unaffected oronly slightly affected by its antimitotic effects. Wewere hesitant to increase the dose of AZA further

due to potential negative side effects of the anti-metabolite drug.

Recent studies have shown much improvedsurvival of xenografted tissue in animals receivinga combination therapy of immunosuppressants ascompared to CsA use alone (Pedersen et al, 1997). Asimilar combination therapy of CsA and MP in ourstudy did not signi®cantly alter the transgeneexpression or immune response. This result issomewhat surprising in light of the many studiesindicating better success with combination drugtherapies. A possible explanation is that CsA is onlypro®cient at prolonging transgene expression at thetargeted site in the CNS at high doses (450 mg/kg/day). We reduced the dose of CsA to 35 mg/kg/dayin combination with MP in accordance with othermultiple-drug protocols for use in allo- and xeno-transplantation studies (Pedersen et al, 1995;Haberal et al, 1995).

It is interesting to note that, although MPsigni®cantly reduced the area of in¯ammationand perivascular in¯ammation proximal to SPNs,it did not signi®cantly prolong transgene expres-sion in SPNs, as did CsA. This ®nding suggeststhat the relationship between the extent ofin¯ammatory cellular in®ltrate and level oftransgene expression by the 14HD3vhsZ is notdirect. Simply reducing the number of immunecells present in the spinal cord does not lead toprolonged transgene expression in SPNs infectedwith the vector. The better effects of CsAtreatment may relate to its actions on cytokineexpression or other factors normally involved inthe immune response to the vector or theencoded transgene. The in vivo effect of CsA oncytokine expression has remained mostly unchar-acterised despite extensive investigation (Shin etal, 1998). The success of CsA in this systemcould be related to its ability to block cytokinesynthesis through blocking calcineurin phospha-tase, an enzyme required in signalling activationof IL-2 and other genes (Daar, 1995). The betterperformance of CsA in this model, moreover, maybe the result of a direct action on the in¯amma-tory macrophages or due to indirect effects likeinhibition of release of substances from in®ltrat-ing T-cells.

Neither CsA nor MP were able to halt theprogression of HSV-1 latency associated termina-tion of the transgene expression in infected SPNs.Thus, although immunosuppression with CsAproved advantageous at early stages by improvingtransgene expression by the vector in SPNs, thiseffect was not long-lived. The eventual loss oftransgene expression may still be overcome throughthe employment of tissue-speci®c promoters usedalone or in conjunction with the HSV-1 latencyassociated promoter (Andersen et al, 1992). Theutilisation of immunosuppression in conjunctionwith vector application, as outlined in this study,

CsA reduces HSV-induced in¯ammation in spinal cordPJ Mabon et al

274

may prove to be useful in determining whichpromoters will enable optimal levels of transgeneexpression.

As part of the immune response, antibodiesdirected against the vector or encoded heterologoustransgene are likely to be produced, althoughappearing late relative to the in¯ammatory process.They may not be playing an active role in reducingexpression of the transgene but would be moreactive following a second challenge with the samevector. This obstacle is signi®cant, as the vector mayrequire repeat administrations to amelioratediseases or neuropathic conditions. To provide¯exibility of repeat administration, immuno-suppression would be necessary to prevent activeremoval of the vector by the host immune system.Further, individuals to be treated may have alreadybeen exposed to HSV-1 and therefore would reactfaster and with a more robust immune response tovector administration, leading to reduction insuccessful therapeutic gene transfer. Thus, animmuno-modulation system in conjunction withan HSV-1 vector system would be vitally importantto increasing the potential success of the genetransfer system. Moreover, immune responses ofthe host directed against viral proteins and/orimmunogenic transgene products expressed bySPNs would potentially limit the persistence ofgene expression (Kaplan and Smith, 1997). Forexample, despite the fact that b-gal is a veryeffective marker enzyme that is expressed earlyafter infection of SPNs and easily detected usingsimple histochemistry, it may be a major factorcontributing to the immunogenicity of the vector.Reports indicate b-gal is likely a potent immunogenresponsible for activating the cellular immuneresponse (Christ et al, 1997; Juillard et al, 1995;Brubaker et al, 1996). Therefore, ®nding an alter-native non-immunogenic reporter gene may be animportant improvement to this vector. Some studieshave reported more successful gene expressionusing host-speci®c marker enzymes. For example,Tripathy et al (1996) found more stable geneexpression in mice using `self' murine erythropoei-tin and only transient gene expression with humanerythropoeitin adenoviral vectors. Potentially ther-apeutic transgenes used in the future also may beimmunogenic.

In this study we did not characterise the cellsinvolved in the immune response to this mod-i®ed, replication-defective triple mutant viralvector. In future studies we will use monoclonalantibodies to identify the cells involved in theimmune response to 14HD3vhsZ or expressedtransgene product, b-gal, in the rat since suchmarkers are not readily available for hamsters. Indoing so, we can gain knowledge regarding thetime course and components of the response anduse this information to develop a more selectiveimmunomodulation strategy aimed at enhancing

the therapeutic potential of this vector for genetransfer.

This study is, to our knowledge, the ®rst to reportimproved ef®cacy of transgene expression in spinalcord neurons infected with a replication defective,triple-mutant HSV-1 vector. This is an importantgene transfer system since it may prove useful fordelivering genes to SPNs and other spinal neuronsthat are targeted most effectively by retrogradetransport from a peripheral administration site.CsA, which has been shown to be a goodimmunosuppressant for transplantation also is agood immunosuppressant for HSV-1-mediated genetransfer. It is likely that each new gene transferprotocol will require experimentation to determinethe best vector dose and mode of immunomodula-tion. Important goals are to achieve strong geneexpression for periods long enough to produce apositive outcome and to permit possible read-ministration of the vector. And, while new genera-tion transduction-ef®cient, non-immunogenicvectors are in development, we can utilise immu-nosuppression with currently available vectors toenable us to express genes of interest and examinethe outcome of this expression in different physio-logical systems.

Materials and methods

Characteristics and preparation of recombinantHSV-1 (14HD3vhsZ)The vector, 14HD3vhsZ, obtained from Dr TheodoreFriedman and Dr Paul Johnson (University ofCalifornia, San Diego, USA) has both copies ofinfected cell protein 4 (ICP4) deleted and thetranscriptional transactivation domain of virusprotein 16 (VP16) inactivated by mutation. Inaddition, the virus host shut-off (vhs) gene has beeninactivated by insertion of the lacZ gene, driven byan immediate early human cytomegalovirus (CMV)promoter (Johnson et al, 1994). Stocks of thereplication defective 14HD3vhsZ were prepared aspreviously described (LeVatte et al, 1998).

Immunosuppression with cyclosporin A (CsA),azathioprine (AZA), or methylprednisolone (MP)From 1 day before adrenal injection with14HD3vhsZ until the time of terminal anaesthesia,hamsters were immunosuppressed with daily in-jections of either CsA (50 mg/kg i.m.; Sandimmune,Novartis), AZA (10 mg/kg, 20 mg/kg i.p.; Imuran,Glaxowelcome), or MP (1 mg/kg or 3 mg/kg i.p.;Solu-Medrol, Upjohn), or a combination of CsA(35 mg/kg i.m.) and MP (3 mg/kg i.p.). Doses of theabove-mentioned drugs were slightly higher thanaverage doses used in xenograft transplantation(Pedersen et al, 1995, 1997; Murase et al, 1993) oraortic allograft arteriosclerosis studies (Lemstrom etal, 1996) in the rat.

CsA reduces HSV-induced in¯ammation in spinal cordPJ Mabon et al

275

Adrenal virus injectionAll protocols for these experiments were approvedby the University of Western Ontario Animal CareCommittee in accordance with the policies estab-lished in the `Guide to the Care and Use ofExperimental Animals' prepared by the CanadianCouncil on Animal Care. All efforts were made tominimize animal suffering and to minimize thenumber of animals used. During the study, allhamsters ate and drank normally and exhibitednormal activity and behaviour. Experiments wereperformed on 31 male Syrian golden hamstersweighing 90 ± 110 g (Charles River, St. Constant,Quebec, Canada). The animals were ®rst sedatedwith diazepam (3.5 mg/kg, i.p.; Sabex Interna-tional Ltd., Boucherville, Quebec, Canada)10 min prior to anaesthesia with sodium pento-barbital (40 mg/kg i.p.; M.T.C. Pharmaceuticals,Cambridge, Ontario, Canada). The virus wasinjected into the left adrenal gland (primary siteof infection) of each animal as previously de-scribed (LeVatte et al, 1997b). We used 6 ml of thevector at 8.756108 plaque forming units per ml(p.f.u./ml). Control animals were injected with14HD3vhsZ in the same way as described pre-viously without accompanying immunosuppres-sant. Animals survived 5 or 8 days. After 5 or 8days, the hamsters were deeply anaesthetised withurethane (3 g/kg i.p.; Aldrich Chemical Company,Inc., Milwaukee, WI, USA), perfused transcar-dially with a 4% formaldehyde ®xation solution.Following perfusion with ®xative, the entirethoracic (T) spinal cord was removed and dividedinto three portions including: T1-5, T6-9, T10-13as described previously (Mabon et al, 1997). Thespinal cord portions were then cut into 30 mm,horizontal sections on a cryostat.

Histochemistry for detection of b-galactosidaseactivityHistochemistry with X-gal (5-bromo-4-chloro-3-in-dolyl-b-D-galactopyranoside) was performed on thehorizontal sections of spinal cord to assay for b-galactivity (Mabon et al, 1997). All sections of spinalcord were mounted onto gelatin coated slides,counterstained with cresyl violet for cytoarchitec-tural analysis using standard procedures, andcoverslipped with DPX mountant (BDH LaboratorySupplies, Poole, UK).

Quantitation of infectionSPNs labelled by X-gal histochemistry to detect14HD3vhsZ were characterised and identi®ed asdescribed previously (Mabon et al, 1997). Uponinjection into the adrenal medulla of the left adrenalgland, 14HD3vhsZ is taken up and transportedretrogradely to cell bodies of adrenal SPNs in theintermediolateral cell column (IML) or lateralfuniculus (LF) of the thoracic spinal cord (Figure1). Sympathetic preganglionic neurons exhibiting b-

gal activity were not evident on the contralateral,uninfected side of the cord. The total number ofSPNs exhibiting b-gal activity was counted in theIML or LF of the thoracic spinal column ipsilateralto adrenal inoculation. The equation of Abercrom-bie was applied to correct for the possibility ofcounting the same neuron more than once (Aber-crombie, 1946). In all analyses, data were collectedunder blinded conditions. A qualitative assessmentof gene expression was done by examining cellmorphology and extent of arborization of dendrites.Greater b-gal activity revealed more dendrites andmore complete ®lling of the neuronal somata withblue reaction product as revealed by X-gal histo-chemistry, whereas less blue reaction product led toreduced elaboration of dendrites and incomplete®lling of somata.

Quantitation of in¯ammatory response to14HD3vhsZAn in¯ammatory in®ltrate occurred around in-fected SPNs in the thoracic spinal cord. To quantifythis in¯ammatory response we used a micro-computer imaging device (MCID) system to tracethe extent of in¯ammation around SPNs andconverted this to an area of in¯ammation (mm2)using preprogrammed calibration standards. Wenormalised these values by dividing the areameasurement by the total number of infected SPNs(detected by X-gal histochemistry) to obtain the areaof in¯ammation per SPN in each animal. Infectedanimals also had evidence of perivascular cuf®ngon the infected side of the spinal cord whereas thecontralateral side had none. We traced areas ofperivascular cuf®ng using the MCID system andsummed the areas per animal. Mean areas werecalculated per treatment group and compared tocontrol values (non-drug treated). In¯ammatoryin®ltrates and perivascular cuf®ng were not ob-served in the corresponding regions of the unin-fected, contralateral side of the spinal cord (data notshown). Statistical analysis of cell counts were doneby the Tukey's t-test after completely randommeasures analysis of variance (Sokol and Rohlf,1981). A probability value of less than 0.05 was usedto indicate statistically signi®cant differences.Values are given as means+standard errors of themean (s.e.m.).

Acknowledgements

This study was supported by the Medical Re-search Council of Canada and the Ontario Heartand Stroke Foundation (OHSF grant T3531). GADis supported by an Ontario Ministry of HealthCareer Scientist Award, and LCW is a CareerInvestigator of the Ontario Heart and StrokeFoundation. The views expressed in this manu-

CsA reduces HSV-induced in¯ammation in spinal cordPJ Mabon et al

276

script are those of the authors and not those of theOntario Ministry of Health. The authors areindebted to Tayfun Atasoy, Aly Cassam, MichaelBygrave and Barbara Atkinson for their technical

assistance. Special thanks are offered to NatalieKrenz, Dr Canio Polosa, and Dr Tony Jevnikar fortheir critical review of this manuscript.

References

Abercrombie M (1946). Estimation of nuclear populationfrom microtome sections. Anat Rec 94: 239 ± 247.

Almawi WY, Beyhum HN, Rahme AA, Rieder MJ (1996).Regulation of cytokine and cytokine receptor expres-sion by glucocorticoids. J Leukoc Biol 60: 563 ± 572.

Andersen JK, Garber DA, Meaney CA, Breake®eld XO(1992). Gene transfer into mammalian central nervoussystem using herpes virus vectors: Extended expres-sion of bacterial lacZ in neurons using the neuron-speci®c enolase promoter. Hum Gene Ther 3: 487 ±499.

Benson A, Ziegler HK (1989). Macrophages as targets forinhibition by cyclosporine. Transplantation 47: 696 ±703

Berkowitz-Balshayi C, Rosen-Wolff A, Darai G, Becker Y(1995). Immunosuppression of Mice by cyclophos-phamide (CyP) and cyclosporin A (CsA) changes thecourse of HSV-1 infection and the distribution of viralDNA in target organs. In Vivo 9: 155 ± 162.

Bloom DC, Maidment NT, Tan A, Dissette VB, FeldmanLT, Stevens J (1995). Long-term expression of areporter gene from latent herpes simplex virus inthe rat hippocampus. Mol Brain Res 31: 48 ± 60.

Bolton C (1995). Recent advances in the pharmacologicalcontrol of experimental allergic encephalomyelitis(EAE) and the implications for multiple sclerosistreatment. Mult Scler 1: 143 ± 149.

Borlongan CV, Stahl CE, Cameron DF, Saporta S,Freeman TB, Cahill DW, Sanberg PR (1996). CNSimmunological modulation of neural graft rejectionand survival. Neurol Res 18: 297 ± 304.

Branisteanu DD, Waer M, Sobis H, Marcelis S, Vande-putte M, Bouillon R (1995). Prevention of murineexperimental allergic encephalomyelitis cooperativeeffects of cyclosporin and 1a,25-(OH)2D3. J Neuroim-munol 61: 151 ± 160.

Brubaker JO, Thompson CM, Morrison LA, Knipe DM,Siber GR, Finberg RW (1996). Th1-Associated im-mune responses to b-galactosidase expressed by areplication-defective herpes simplex virus. J Immunol157: 1598 ± 1604.

Calder VL, Bellamy AS, Owen S, Lewis C, Rudge P,Davidson AN, Feldmann M (1987). Effects of cyclos-porin A on expression of IL-2 and IL-2 receptors innormal and multiple sclerosis patients. Clin ExpImmunol 70: 570 ± 577.

Chaudhary LR, Avioli LV (1996). Regulation of inter-leukin-8 gene expression by interleukin-1 beta,osteotropic hormones, and protein kinase inhibitorsin normal human bone marrow stromal cells. J BiolChem 271: 16591 ± 16596.

Christ M, Lusky M, Stoeckel F, Dreyer D, Dieterle A,Michou AI, Pavirani A, Mehtali M (1997). Genetherapy with recombinant adenovirus vectors: evalua-tion of the host immune response. Immunol Lett 57:19 ± 25.

Daar AS (1995). Developments in immunosuppressivetherapy. Transplant Proc 27: 2671 ± 2675.

Davidson BL, Allen ED, Kozarsky KF, Wilson JM,Roessler BJ (1993). A model system for in vivo genetransfer into the central nervous system using anadenoviral vector. Nat Genet 3: 219 ± 223.

Davidson BL, Bohn MC (1997). Recombinant adenovirus:A gene transfer vector for study and treatment of CNSdiseases. Exp Neurol 144: 125 ± 130.

Durham HD, Alonso-Vanegas MA, Sadikot AF, Zhu L,Lochmuller H, Massie B, Nalbantoglu J, Karpati G(1997). The immunosuppressant FK506 prolongstransgene expression in brain following adenovirus-mediated gene transfer. NeuroReport 8: 2111 ± 2115.

Fink DJ, DeLuca NA, Goins WF, Glorioso JC (1996). Genetransfer to neurons using herpes simplex virus-basedvectors. Ann Rev Neurosci 19: 265 ± 287.

Frishberg Y, Meyers CM, Kelly CJ (1996). Cyclosporine Aregulates T cell-epithelial cell adhesion by alteringLFA-1 and ICAM-1 expression. Kidney Int 50: 45 ± 53.

Glorioso JC, Bender MA, Goins WF, Fink DJ, DeLuca N(1995a). HSV as a gene transfer vector for the nervoussystem. Molec Biotech 4: 87 ± 98.

Glorioso JC, DeLuca NA, Fink DJ (1995b). Developmentand application of herpes simplex virus vectors forhuman gene therapy. Ann Rev Microbiol 49: 675 ±710.

Glorioso JC, Goins WF, Meaney CA, Fink DJ, DeLuca NA(1994). Gene transfer to brain using herpes simplexvirus vectors. Ann Neurol 35: S28 ± S34.

Guerette B, Vilquin JT, Gingras M, Gravel C, Wood KJ,Tremblay JP (1995). Prevention of immune reactionstriggered by ®rst-generation adenoviral vectors bymonoclonal antibodies and CTLA4Ig. Human GeneTher 7: 1455 ± 1463.

Haberal M, Demirag A, Bilgin N, Arslan G, Buyukpa-mukcu N (1995). Immunosuppressive Protocols atTransplantation Centers in Turkey. Transplant Proc27: 2776 ± 2777.

Hermens WTJMC, Verhaagen J (1997). Adenoviral vector-mediated gene expression in the nervous system ofimmunocompetent wistar and T cell-de®cient nuderats: preferential survival of transduced astroglial cellsin nude rats. Human Gene Ther 8: 1049 ± 1063.

Herz J, Gerard RD (1993). Adenovirus-mediated transferof low density lipoprotein receptor gene acutelyaccelerates cholesterol clearance in normal mice. ProcNatl Acad Sci USA 90: 2812 ± 2816.

Hong S, Neve R, Howe J, Kalb RG (1995). Gene transferinto spinal cord using HSV vectors. Neurosci Abs 21:536 (Abstract).

Huard J, Akkaraju G, Watkins SC, Pike-Cavalcoli M,Glorioso JC (1997). LacZ gene transfer to skeletalmuscle using a replication defective herpes simplexvirus type 1 mutant vector. Human Gene Ther 8:439 ± 452.

CsA reduces HSV-induced in¯ammation in spinal cordPJ Mabon et al

277

Huitinga I, Ruuls SR, Jung S, Vanrooijen N, Hartung H-P,Dijkstra CD (1995). Macrophages in T cell line-mediated, demyelinating, and chronic relapsing ex-perimental autoimmune encephalomyelitis in Lewisrats. Clin Exp Immunol 100: 344 ± 351.

Johnson PA, Wang MJ, Friedman T (1994). Improved cellsurvival by the reduction of immediate-early geneexpression in replication-defective mutants of herpessimplex virus type 1 but not by mutation of the virionhost shutoff function. J Virol 68: 6347 ± 6362.

Juillard V, Villefrox P, Godfrin D, Pavirani A, Venet A,Guillet JG (1995). Long-term humoral and cellularimmunity induced by a single immunization withreplication-defective adenovirus recombinant vector.Eur J Immunol 25: 3467 ± 3473.

Kaplan JM, Smith AE (1997). Transient immunosuppres-sion with deoxyspergualin improves longevity oftransgene expression and ability to readministeradenoviral vector to the mouse lung. Human GeneTher 8: 1095 ± 1104.

Kasaian MT, Biron CA (1990). Effects of cyclosporin Aon IL-2 production and lymphocyte proliferationduring infection of mice with lymphocytic choriome-ningitis virus. J Immunol 144: 299 ± 306.

Keir SD, Mitchell WJ, Feldman LT, Martin JR (1995).Targeting and gene expression in spinal cord motorneurons following intramuscular inoculation of anHSV-1 vector. J Neurovirol 1: 259 ± 267.

Kennedy PGE, Steiner I (1993). The use of herpessimplex virus vectors for gene therapy in neurologicaldisease. Quart J Medicine 86: 697 ± 702.

Lemstrom KB, Raisannen-Sokolowski AK, Hayry PJ,Koskinen PK (1996). Triple Drug ImmunosuppressionSigni®cantly Reduces Aortic Allograft Arteriosclerosisin the Rat. Arteriosclerosis, Thrombosis, and VascularBiology 16: 553 ± 564.

LeVatte MA, Cassam AK, Dekaban GA, Weaver LC(1998). Analysis of a multi-mutant herpes simplexvirus type 1 for gene transfer into sympatheticpreganglionic neurons and a comparison to adeno-virus vectors. Neurosci 86: 1321 ± 1336.

LeVatte MA, Dekaban GA, Weaver LC (1997a). Genetransfer into sympathetic preganglionic neurons invivo using a nonreplicating thymidine kinase de®-cient herpes simplex virus type 1. Neurosci 80: 893 ±906.

LeVatte MA, Mabon PJ, Weaver LC, Dekaban GA(1997b). Simultaneous identi®cation of two popula-tions of sympathetic preganglionic neurons usingrecombinant herpes simplex type 1 virus expressingdifferent reporter genes. Neurosci 82: 1253 ± 1267.

Liu T, Tang Q, Hendricks RL (1996). In¯ammatoryin®ltration of the trigeminal ganglion after herpessimplex virus type 1 corneal infection. J Virol 70:264 ± 271.

Mabon PJ, LeVatte MA, Dekaban GA, Weaver LC (1997).Identi®cation of sympathetic preganglionic neuronscontrolling the small intestine in hamsters using arecombinant herpes simplex virus type-1. Brain Res753: 245 ± 250.

Martin D, Near SL, Bendele A, Russell DA (1995).Inhibition of tumor necrosis factor is protectiveagainst neurologic dysfunction after active immuniza-tion of Lewis rats with myelin basic protein. ExpNeurol 131: 222 ± 228.

Murase N, Demetris AJ, Tanabe M, Miyazawa H,Valdivia LA, Nakamura K, Starzl TE (1993). Effect ofFK506 and antiproliferative agents for heart and liverxenotransplantation from hamster to rat. TransplantProc 25: 425 ± 426.

Mustafa M, Diener P, Sun J-B, Link H, Olsson T (1993).Immunopharmacologic modulation of experimentalallergic encephalomyelitis: low-dose cyclosporin Atreatment causes disease relapse and increasedsystemic T and B cell-mediated myelin-directedautoimmunity. Scand J Immunol 38: 499 ± 507.

Palay DA, Cluff CW, Wentworth PA, Ziegler HK (1986).Cyclosporine inhibits macrophage-mediated antigenpresentation. J Immunol 136: 4348 ± 4353.

Pedersen EB, Poulsen FR, Zimmer J (1995). Prevention ofmouse-rat brain xenograft rejection by a combinationof cyclosporin A, prednisolone and azathioprine. ExpBrain Res 106: 181 ± 186.

Pedersen EB, Zimmer J, Finsen B (1997). Tripleimmunosuppression protects murine intracerebral,hippocampal xenografts in adult rat hosts: effects oncellular in®ltration, major histocompatability complexantigen induction and blood-brain barrier leakage.Neurosci 78: 685 ± 701.

Peltekian E, Parrish E, Bouchard C, Peschanski M,Lisovoski F (1997). Adenovirus-mediated gene trans-fer to the brain: methodological assessment. J Neu-rosci Meth 71: 77 ± 84.

Perico N, Remuzzi G (1997). Prevention of transplantrejection; current treatment guidelines and futuredevelopments. Drugs 54: 533 ± 570.

Pyner S, Coote JH (1994). A comparison between theadult rat and neonate rat of the architecture ofsympathetic preganglionic neurones projecting to thesuperior cervical ganglion, stellate ganglion andadrenal medulla. J Auton Nerv Syst 48: 153 ± 166.

Schramm LP, Adair JR, Stribling JM, Gray LP (1975).Preganglionic innervation of the adrenal gland of therat: a study using horseradish peroxidase. Exp Neurol49: 540 ± 553.

Shin GT, Khanna A, Ding R, Sharma VK, Lagman M, LiB, Suthanthiran M (1998). In vivo expression oftransforming growth factor-beta in humans ± Stimula-tion by cyclosporine. Transplantation 65: 313 ± 318.

Sokol RR, Rohlf FJ (1981). Biometry. In: The Principlesand Practice of Statistics in Biological Research. WHFreeman and Co., San Francisco. pp 175 ± 204.

Tripathy SK, Black HB, Goldwasser E, Leiden JM (1996).Immune responses to transgene-encoded proteinslimit the stability of gene expression after injectionof replication-defective adenovirus vectors. Nat Med2: 545 ± 550.

Wolfe JH, Deshmane SL, Fraser NW (1996). Herpes virusvector gene transfer and expression of B-glucuroni-dase in the central nervous system of MPS VII mice.Nat Genet 1: 379 ± 384.

Wood MJA, Byrnes AP, Pfaff DW, Rabkin SD, CharltonHM (1994). In¯ammatory effects of gene transfer intothe CNS with defective HSV-1 vectors. Gene Therapy1: 283 ± 291.

Wu L, Morahan PS, Leary K (1993). Regulation of herpessimplex virus type 1 gene expression in nonpermis-sive murine resident peritoneal macrophages. JLeukoc Biol 53: 61 ± 65.

CsA reduces HSV-induced in¯ammation in spinal cordPJ Mabon et al

278

Yang Y, Nunes FA, Berenesi K, Furth EE, Gonczol E,Wilson JM (1994). Cellular immunity to viral antigenslimits E1-deleted adenoviruses for gene therapy. ProcNatl Acad Sci USA 91: 4407 ± 4411.

Zamvil SS, Steinman L (1990). The T lymphocyte inexperimental allergic encephalomyelitis. Ann RevImmunol 8: 579 ± 621.

CsA reduces HSV-induced in¯ammation in spinal cordPJ Mabon et al

279