retroviruses and xenotransplantation: determining the risks

Toxic triplets provideclues toneurodegenerationCleavage, aggregation and toxicity ofthe expanded androgen receptor inspinal and bulbar muscular atrophy

Merry, D.E. et al. (1998)

Hum. Mol. Genet. 7, 693–701

Truncated N-terminal fragments ofhuntingtin with expanded glutaminerepeats form nuclear and cytoplasmicaggregates in cell culture

Cooper, J.K. et al. (1998)

Hum. Mol. Genet. 7, 783–790

Aggregation of N-terminal huntingtin isdependent on the length of itsglutamine repeats

Li, S.H. and Li, X.J. (1998)

Hum. Mol. Genet. 7, 777–782

Spinal and bulbar muscular atrophy (SBMA) andHuntington’s disease (HD) are among the neuro-degenerative disorders characterized by expandedsegments of glutamine-encoding CAG trinucleotiderepeats in the protein-coding regions of the affectedgenes. Recent studies in transgenic animals havedemonstrated that mice overexpressing truncatedforms of these proteins (including expanded repeats)develop neurodegenerative disorders that are, inpart, similar to the human diseases. How expressionof these abnormal proteins selectively kills neuronsremains unknown, although the formation of abnor-mally folded aggregates of the proteins, particularlyin the nuclei of neurons, might play a role.

The genes responsible for SBMA (androgen re-ceptor) and HD (huntingtin) have now been ex-pressed in cell lines, with varying degrees of suc-cess at examining toxicity of their gene products.Merry et al. expressed constructs containing a por-tion of the androgen receptor gene with 16–112 glut-amines in COS-7 (transformed green monkey kid-ney fibroblasts) and MN-1 (hybrid cells derived fromembryonic mouse spinal cord neurons) cell lines.Constructs containing more than 65 repeats weremore likely to form aggregates (detected both byimmunoblotting of extracted proteins and by im-munocytochemistry); the number of cells with ag-

gregates increased with the number of trinucleotiderepeats. Both nuclear and cytoplasmic aggregateswere seen; nuclear aggregates were more frequentin the neuronal cell line. Toxicity was examined onlyin the COS-7 cells, and although more cells trans-fected with the constructs containing more than 65repeats died than did control cells transfected witha b-galactosidase-encoding plasmid, overall levelsof death were very low (a maximal 6% of trans-fected cells were dead 48 h after transfection).

Cooper et al. and Li and Li obtained similar resultsusing transfection of truncated huntingtin constructsinto fibroblast, neuroblastoma and PC12 cell lines.Aggregates were formed when constructs containedmore than 70–80 glutamine repeats; more aggre-gates formed with longer repeats and with constructsthat yielded a more-truncated huntingtin protein.Cooper et al. found no toxicity of the huntingtin con-structs alone in neuroblastoma cells, although cellstransfected with a truncated huntingtin construct con-taining 82 repeats were nearly twice as vulnerable tostaurosporine exposure as cells transfected with aconstruct containing only 19 glutamine repeats.

These studies provide a new approach for under-standing neuronal death in triplet repeat disorders, andraise the possibility that expression of proteins contain-ing expanded repeats, while not directly toxic, might enhance the susceptibility of neurons to other insults.

B. Joy Snider MD, PhDInstructor in Neurology, Dept of Neurology, Center forthe Study of Nervous System Injury, WashingtonUniversity School of Medicine, Box 811, 660 S. EuclidAvenue, St Louis, MO 63110-1093, USA.

Retroviruses andxenotransplantation:determining the risksIdentification of a full length cDNA for anendogenous retrovirus of miniature swine

Akiyoshi, D.E. et al. (1998)

J. Virol. 72, 4503–4507

Type C retrovirus released from porcineprimary peripheral blood mononuclearcells infects human cells

Wilson, A.C. et al. (1998)

J. Virol. 72, 3082–3087

The number of patients requiring organ transplant-ation far outweighs the availability of suitable

human donor organs. This is an acute problemthat is exacerbated by an ageing population, andxenotransplantation of pig organs is gainingfavour as a solution to this shortage. While pro-gress has been made on the immunological prob-lems of xenotransplantation, the risk of infectiousdisease transmission from graft to recipient, andconceivably onto the new host population, is anunknown quantity underpinned by relatively littleresearch. On the one hand, the known infectiouspathogens of pigs can be monitored and largelyeliminated through the breeding of specificpathogen-free herds. On the other, the existenceof unknown, often latent, infections that are asymp-tomatic in their natural host, but that could causedisease in the xenografted recipient, are of principalconcern. Porcine retroviruses fall into this categoryand consequently are receiving increasing attention.

The life-cycle of retroviruses involves the inte-gration of the viral genome into host chromatin, tocreate a so-called provirus. This is a permanentassociation between the cell and retrovirus, andprovides the means for the retrovirus to colonizethe germline of its host, where it can persist as astable, integrated provirus for multiple gener-ations. The normal germline DNA of all vertebratespecies, including pigs and humans, containssuch endogenous retroviruses (ERVs), as well asdefective retroviral sequences called retroel-ements. Even though ERVs are not normally patho-genic in their natural host species, the possibilitythat these might cause disease if transmittedthrough xenotransplantation is a significant worry.

In the first of these papers, Akiyoshi et al. haveprovided the first full-length sequence of a porcineERV genome. The sequence information avail-able on other porcine ERVs is limited, although theavailable comparisons demonstrate strong hom-ology. This work will allow the design of tools fordetecting other porcine retroviruses, as well asproviding the basis of a deeper understanding oftransmission and viral activation. In the secondpaper, Wilson et al. have identified mitogenic con-ditions for the activation of a porcine ERV and sub-sequent release of retrovirus particles from porcineperipheral mononuclear blood cells. Significantly,this retrovirus can infect human cells in vitro andthis work provides the first demonstration thatsuch an infectious virus can be mobilized from pri-mary porcine cells. Previously, ERVs capable ofinfecting human cells have been reported from es-tablished porcine cell lines, so it is not surprisingthat further evidence of ERV zoonosis is mounting.

Although it is difficult to predict the clinical out-come of human infection by porcine ERVs, similarretroviruses have been associated with disease innon-human primates. Indeed, a particularly poignantstudy for xenotransplantation has been the devel-opment of lymphoma in immunosuppressed

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non-human primates after exposure to replicatingretroviruses. Furthermore, the recombinogenicproperties of retroviral sequences might offer thepossibility of creating a hybrid retrovirus betweenthe incoming porcine retrovirus and silent retrose-quences or ERVs in the human genome.Implications for the emergence of a new type ofretroviral pathogen are already documented bythe story of HIV, and this underscores the impor-tant considerations in assessing the safety risksassociated with xenotransplantation.

Roger Hewson PhDCentre for Applied Microbiology and Research, PortonDown, Salisbury, Wiltshire, UK SP4 0JG.

New evidence forneuroprotection byestrogenEstrogen reduces neuronal generationof Alzheimer b-amyloid peptides

Xu, H. et al. (1998)

Nat. Med. 4, 447–451

Nuclear estrogen receptor-independentneuroprotection by estratrienes: a novelinteraction with glutathione

Green, P.S. et al. (1998)

Neuroscience 84, 7–10

Recent epidemiological and clinical studies sug-gest that estrogen-replacement therapy mightprotect postmenopausal women from Alzheimer’sdisease and improve their cognitive function. Thesubstantial potential for prevention and therapyof this devastating disease by estrogen hormonesis now beginning to emerge. Estrogen’s Alzheimer-busting potential should be enhanced by discern-ing the mechanisms underlying its neuroprotec-tive properties. Previous studies have demon-strated that treating cultured neuronal cells with17b-estradiol, the physiological estrogen hor-mone, increased the secretion to the culturemedium of b-APP, the membrane-spanning pre-cursor for b-amyloid peptides, which are the majorcomponent of the amyloid plaques found in post-mortem brains of individuals with Alzheimer’s dis-ease. Because secreted b-APP, unlike full-lengthmembrane b-APP, cannot serve as a precursorfor b-amyloid peptides, researchers reasonedthat increasing secretion of soluble b-APP should

reduce the yield of b-amyloid peptides in the agingbrain. An important lead in this direction has nowbeen supplied by Xu et al., who showed thattreating cultured neuronal cells with 17b-estradioldiminishes the production of b-amyloid peptidesto almost half the control levels. This effect waslong-lasting, as it was observed in cultures treatedwith physiological concentrations of 17b-estradiolfor periods of several weeks. This observation isparticularly encouraging compared with previousin vitro strategies, in which increased soluble b-APP secretion or decreased b-amyloid produc-tion usually lasted only a few hours to a few days.

Other putative neuroprotective effects of estro-gen hormones might include modulation of basalforebrain cholinergic activity and prevention ofoxidative stress and b-amyloid-mediated toxicity.Green et al. have now shown that the latter effectis unrelated to the known nuclear estrogen receptor: treatment of HT-22 murine neuronalcells with either 17b-estradiol, 17a-estradiol, orestratrien-3-ol protected them from b-amyloidtoxicity. Notably, adding reduced glutathione to theculture medium increases the neuroprotectivepotency of estrogens by about 400-fold, suggestingan antioxidant-related mechanism of neuropro-tection. Together, the new studies raise hopes fora combined estrogen/antioxidant therapy forAlzheimer’s disease, and possibly for other neuro-degenerative diseases, in the future.

David Gurwitz PhD (gurwitz@post.tau.ac.il)National Laboratory for the Genetics of IsraeliPopulations, Sackler Faculty of Medicine, Tel-AvivUniversity, Tel-Aviv 69978, Israel.

Winning the war againstantibiotic resistanceAntibiotic sensitization using biphenyltetrazoles as potent inhibitors ofBacteroides fragilis metallo-b-lactamase

Toney, J.H. et al. (1998)

Chem. Biol. 5, 185–196

Autoinducer of virulence as a target forvaccine and therapy againstStaphylococcus aureus

Balaban, N. et al. (1998)

Science 280, 438–440

Multiple antibiotic resistance among bacteria is becoming an increasingly alarming problem for the

clinician. Even the antibiotic of last resort, vanco-mycin, is no longer all conquering [see, for example,Healy, V.L. et al. (1998) Chem. Biol. 5, 197–207]. Inthese papers, two approaches to getting around thisproblem are described.

Species of Bacteroides, including B. fragilis,are commonly found in clinical isolates from suppu-rative or surgical infections. Simultaneous resistanceto metronidazole, co-amoxiclav and imipenem in asingle clinical isolate of B. fragilis was recentlyfound to be due to a metallo-b-lactamase-like en-zyme produced by the bacteria. Toney et al. set outto determine whether combination therapy withantibiotics and inhibitors of metallo-b-lactamasesmight revive the activity of the antibiotics. Byscreening of a large chemical library and modellingbased on X-ray crystallography, the authors identi-fied a series of biphenyl tetrazoles (BPTs) that in-hibited b-lactamase activity. In a simple assay ofantibiotic activity, in which zones of growth inhibi-tion on lawns of bacteria were measured, theywere able to show synergistic inhibition by a BPTand imipenem – thus converting a clinically re-sistant strain of B. fragilis to a sensitive one.

In a strikingly different approach, Balaban et al.have targeted the production of bacterial toxins byStaphylococcus aureus. The synthesis of virulencefactors in S. aureus is controlled by the regulatoryRNAmolecule RNAIII, which is itself auto-induced bythe RNAIII-activating protein (RAP). In a mousemodel, the authors showed that vaccination of theanimals with purified RAP had the effect of dramati-cally reducing both the mortality and morbidity as-sociated with the S. aureus infection. Only one out of33 vaccinated mice died, whereas five of 22 untreatedanimals died. The lesions in surviving mice weremuch smaller in the vaccinated group than those inthe controls. In addition, a non-pathogenic strain ofS. aureuswas found to produce a peptide, YSPXTNF,that inhibited RAP and has therefore been calledRIP. Injection of purified RIPand S. aureus protectedmice against a sub-lethal dose of the bacterium.Further good news is that regulatory mechanisms in-volving auto-inducers have been described for otherbacterial species.

Together, these papers give hope to the clini-cian that the war against antibiotic-resistant bacteria is far from lost.

Ian A. McKay MA, PhDSenior Lecturer in Dermatology, Centre for CutaneousResearch, 2 Newark Street, London, UK E1 2AT.

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The literature page is designed to alert readers to recent pri-mary papers that are relevant to molecular medicine. Apanel of senior active researchers is commissioned to se-lect one or two recent papers within their area of expertise.If you are interested in joining this panel, send an e-mailmessage to the Editor at: mmt.trends@elsevier.co.uk