n the early 1970s immunol~ogists at

the Weizmann Institute of Science,

Rehovot, Israel, described the phe-

nomenon of oxidative mitogen-

e&r. In cultures containing accessory cells

and lymphocytes, they showed that chemi-

cal or enzymatic generation of reactive alde-

hydes at the cell surface resulted in lym-

phocyte activation, an effect subsequently

confirmed by others3s4. In 1988, using poly-

meric aldehyde-treatment of antigen pre-

senting cells (APCs), initially to study the

role of cytokines in specific T-cell activation,

very low-dose (but not high-dose) aldehyde

treatment of APCs was found to abolish

antigen (Ag)-dependent T-cell activation5.

The brief 30 s reaction between the very low

concentrations of polymeric aldehyde emplo, vet

Cosfimulatory macromolecules on

antigen-presenting cells nnd T cells

play a critical regulfltory role in

immune induction and thus make

useff41 immunotkerapeufic targets.

Huzoevcr, ns reviewed here, by

John Rhodes, studies of

complementary chemical events are

beginning to reveal a new level of

intercellular and intracellular

signalling that can be targeted by

small, orally active therapeutic

agents. One such molecule,

tucaresol, is being developed for

testing ns n systemic

immunopofenfiatory drug.

render them Irreversible; (4) covalent cell-

surface labelling methods to visualize con-

stitutive Schiff-base-forming ligands by

flow cytofluorimetric and immunohisto-

chemical techniques. The formation of

Schiff bases between ARC and T-cell is illus-

trated schematically in Fig. 1.

i dnd ARC surface

amino groups does not fix APCs (although many incorrectly refer to

these cells as ‘lightly fixed). Instead the reaction results in the co-

valent ligation of a small, highly reactive minority of surface E-

amino groups in an irreversible Schiff-base linkage. This suggested

that Schiff-base formation, pre-empted by the chemical treatment,

might be playing an essential role in AK-T-cell interactions, and

that oxidative mitogenesis, which generates cell-surface Schiff-base-

forming aldehydes, might be an experimental amplification of a

normal physiological process. Although this mechanism is un-

precedented in physiological interactions between cells, Schiff-base

formation is an ancient and widely distributed mechanism in signal

transduction processes and enzyme+ubstrate interactions (see Box 1).

In subsequent studies, this hypothesis was tested in in vitro mod-

els of T-cell responsiveness, using the chemical criteria defining

S&ii-base formation. Invariably, the r&uh.- were consistent with

intercellular Schiff-base formation betweco surface carbonyls and

amines playing an essential role in ARC-T-cell interactions’“17. The

Initial evidence for this model of events depended on the following

criteria: (1) chemically-defined modifications of cell-surface Schiff-

base-forming ligands to block inductive AK-T-cell interactions; (2)

chemical and enzymatic re-donation of Schiff-base-forming ligands

to restore ARC-T-cell interactions; (3) the use of the selective reduc-

ing agent cyanoborohydride to specifically label Schiff bases and

The next step was to realize that if oxida-

tive mitogenesis were amplifying a normal

event in Ag-dependent T-cell activation,

then it might be possible to exploit aldehyde-

generating agents as novel immunologicaI

adjuvants. This proved to be the case with

neuraminidase-galactose oxidase (NAGO),

which generates aldehydes on carbon 6 of

cell-surface galactose (Fig. Z)*. Surprisingly

(in view of its broad polyclonal effects in vitro), NAG0 acted as a potent immunological adjuvant for specific

responses to bacterial, viral and protozoa1 subunit vaccines in mice

and was especially effective in the induction of cytotoxic T lym-

phocyte (CTL) responses Is. NAG0 also yielded some highly en-

couraging results in d pilot study of bovine vaccination with native

glutathione-S-transferase from Fascioh heputicd9. However, the pro-

tection results were difficult to reproduce in further bovine trials,

possibly reflecting a particular susceptibility of this immunogen

to batch variation when formulated in this manner (C. Morrison,

unpublished).

Fundamental and therapeutic progress was made when it was

discovered that small exogenous Schti-base-forming molecules

could substitute for the natural donor of carbonyl groups and pro-

vide a costimulatory signal to CD4+ T helper (Th) cell+. These mol-

ecules could therefore act as biochemical probes to investigate both

the costimulatory signalhng pathway initiated by Schiff-base for-

mation at the T-cell surface, and the signai-transducing macromol-

ecules involved; they also were candidate molecules for testing as

orally delivered, systemically active costimulatory drugs. A key to

pursuing this drug development strategy was the knowledge that

small Schiff-base-forming drugs had already been developed for

another purpose - to modify haemoglobm in sickle cell anaemia”.

Clearly it was possible to obtain a favourable profile in terms of

pharmacokinetics and systemic tolerability with such molecules.

~- IMMUNOLOGY TODAY

hlU_ Constitutive

Fig. I. As a consequence of certain macromolecular interactions during

APC-T-cell conjugation, transient covalent chemical events take place in lhr form of Schiff-base formation between specinlized carbonyls and

amines. This process, which is rapid, dynamic and highly reversible, ap- pears to be essential for optimal T-cell activation and provides a targef for

the manipulation of immune responses. Abbreviations: AK, antigen- presenting cell; MHC, major histocompatibility complex; TCR, T-cell receptor; Th, T helper.

However, it turned out that one such drug was found to be the best

candidate as an orally delivered, systemically active immuno-

potentiator with effects on the immune system occurring at much

lower doses than those required to modify haemoglobin. The drug

of choice is a substituted benzaldehdye, 4[2-formyl-3-hydroxy-

phenoxymethyl) benzoic acid, named tucaresol”.

Effects of Schiff-base costimulation by tucaresol on the immune response The small xenobiotic molecule tucaresol forms Schiff bases on

T-cell-surface amines through its aldehyde (see Fig. 3) and the reac-

tion can be visualized by competitive covalent ligation with sulpho-

N-hydroxysuccinimidobiotin (SNHS-biotin). Experimentally cell-

surface amines are allowed to react with tucaresol and the resultant

Schiff base is subsequently rendered irreversible by using the selec-

tive reducing agent, cyanoborohydride. This reaction is then meas-

ured by the loss of free cell-surface ammo groups available to react

with SNHS-biotin in a flow cytofluorimetric assay”. Schiff-base

formation by tucaresol on cell-surface amines, proceeding within

seconds, can also be measured by spectrophotometric methods as

well as by radiolabelling with cyanoborotritiide or radiolabelled drug.

As a result of this covalent chemical event, the CD4+ T cell re-

ceives a costimulatory signal. Optimally, a five- to tenfold enhance-

ment of interleukin 2 (IL-Z) production is observed when the T cell

is stimulated with anti-CD3 in the presence of tucaresol. In general,

tucaresol favours a Thl-type profile of cytokine production, en-

hancing the production of IL-2 and interferon y (IFN-$ but not IL-4

or IL-6 (Ref. 11). The current picture of Thl- and Th2-type responses

suggests that this is likely to be favourable in immunopotentiatory

therapy for intracellular pathogens such as viruses, mycobacteria

and protozoa1 parasites, as well as immunogenic tumour~“-~.

BOX I. Skhiff-base formation in phyIidogi4 systems

h chemical terms, Schiff-base formation is a carhnyl_amino

condensation that occurs as a result of nucleophllic attack by the

amine on the electron deficient carbonyl group in the following reaction:

R&=0 + NH& -+ R,-C=N-R, + I-&,0

The term ‘Schiff base’ is properly applied when the nitrogen

of the amine is attached to a carbon. If it is attached to another

nitrogen then the condensation product is a hydrazone rather

than a Schiff base. The initial reaction forms an ambmcmbmol

that is usually unstable and either reverts to the starting mate+

als or dehydrates to the Schiff base (imine). In general terms the

imine or Schiff-base product is itself a reactive s@es and is

prone to further reactions resulting in the addition of m&o-

phibc agents (e.g. HJVR, HSR and HOR) to the imine bond6.

S&i&base formation is essential to a number of dynamic

physiological processes, including enzyme-substrate interac-

tion~ such a~ those mediated by pyrldoxal phosphate, muscle

aldolase, lysyi oxidase and acetoacetate decarboxylaser. In all

cases where M-base formation is important in a physiological

process, the carbonyl donor is an organic prosthetic group or

coenzyme such as pyridoxal phosphates. In lysyl oxidase the car-

bony1 donor is the di-ketone moiety of pynoloquinoline

quinone9. For example, in the vertebrate visual system, Schiff-

base formation between retinaldehyde (vitamin A aldehyde) and

rhodopsin is central to the process of changing light energy into

a neuralsignal in the form of Na+ flm~~~. Light falling on 11 cis-

retinal produces a conformational change that results in a 3 A shift in the W-base linkage in relation to the ring structure of

the chromophom. This produces a conformational change in

rhodopsin that activates a Gprotein, transducin, which in turn

links the activation event to a phosphodiesterase which hydro-

lyses cyclic GMP (CCMP). The fall in cGh4P levels then closes Nat

channels in the rod plasma membrane, resulting in hyperpolar-

ization. Schiff-base form&ion by tucareso 1 on nudeophihc

amines on the plasma membrane of T cells has marked effects on

Na+ and K+ flux (see below) providing an interesting parallel

with the visual system**. Schtff-base fmnation is ah at

the centre of ltghtdriven ion pumps in bacteria”. These seven-

tmn.smembranedomain (rhodopsin-like) receptors and their

durnnophores consist of retinal linked to a lyslne residue by

means of a protonated Schlff base near the middle of helix G. IA)

bacteriorhodopsin, proton trim&r from the retinal Schiffbase to

aspartame 85 is crucial in the transport cycle. RemarkaMy, it has

n~ent~ been shown that substituting the bacteriorhodopsln

aspartate 85 for the halorhodopsin thmonine 85 funaionally

tonwrts bacteriorhodopsin (proton import) into halorhodopsin (doride export) when expressed in Ha&bactf?riU~ scl~iWiU~‘3.

Covdent chemical events in the immune system might evemu-

ally turn out to sham some of the characteristics of light-driven

I& other Schlff-base-dependent mechanisms already Well-

&aract&zed in enzyme catalysis and signal transduction.

SEPTEMBER 1996

ReceDtor . NH2

Fig. 1. A combination of the two enzymes neuraminidase and galactose oxidase (NAGO) greatly amplifies the expression of cell-surface Schifl-base-forming carbokyls by exposing galsctose and gen- erating an aldehyde on carbon-6 (shown in red). These enzymes potently enhance immune responses to vaccine antigens in mice zohen administered locally as an adjuvant. iI seems likely that thebnc-

tionally important neo-aldehydes are on the macromolecules fhaf express constitutive carbonyl groups. For abbreviations, see Fig. 1 legend.

OH

CHO

0-CH2 COOH

Tucaresol

Fig. 3. The substituted benzaldehyde, 4(2-formyl-3-hydroxy-oxy-phecoxy-

methyl) benzoic acid or tucaresol, is an orally delivered, systemically ac- tive immunopotentiatoy drug that zvorks through its covalent reacti&y

with funcConally specialized amino groups at the surface of T cells. Its re- activity is through the aldehydefunction (shozun in red), which condenses with amines to form a readily reversible Schiff base.

These effects in vitro are fully reflected in vivo: indeed the drug is

considerably more potent in vivo where low concentrations produce

very substantial effects in the presence of antigen. Tucaresol given

parenterally or orally, at an optimum dose, potently enhances both

the CD4+ Th-cell response to administered antigens and the gener-

ation of CD8’ CTL responses, and is therapeutically effective in

murine models of virus infection and tumour growth”. In an acute

model of cytomegalovirus infection tucaresol substantially reduces

viral load over a five-day course of treatment”. In a model of syn-

geneic tumour growth, tucaresol was found to be effective in reduc-

ing fhe outgrowth of the weakly immunogenic ader3carcinoma,

MCA38 (Ref. 11).

Despite the development of potent Schiff-base-forming drugs

with broad therapeutic potential, the Schiff-base mechanism in

APC-T-cell inductive interactions is a new area of investigation

with many questions remaining to be answered. The extent oi its

physiological importance and explanatory value will emerge only

with the definitive identification of the natural donor of carbnyi

groups, its macromolecular targets, and the

signalling pathway it employs.

However, many frequently expressed

reservations regarding the mechanism can

already be answered as follows. First, the

availability of Schiff-base-forming carbonyl

groups appears to be limited in view of the

rarity of these groups in glycoproteins.

Nevertheless, constitutive carbonyl groups

can readily be demonstrated on lymphoid

cells by covb!enf labelling with biotin hy-

drazide’s. These groups are not thought to

be on glyceproteins but on an organic

prosthetic group noncovalently bound to

specialized proteins. Second, interactions

between cells of the immune system are

transient and of a relatively weak affinity

apparently incompatible with covalent link-

ages. Schiff-base formation, however, is a

transient, highly reversible reaction occur-

ring over seconds in physiological settings

(such as the visual system!. Rapid reversal of

the Schiff-base linkage is an integral part of these processes. Third,

formation of covalent links between constitutive groupa appears to

be at odds with the specificity of immune responses. However,

Schiff-base formation is envisaged to occur only as a consequence of

conventional stereospecific interactions between accessory macro-

molecules, excluding random or promiscuous reactions.

Because Schiff-base-forming drugs are believed to work by mim-

icking the natural donor of Schiff-base-forming carbony!s, they can

also be exploited as biochemical probes to investigate the costimu-

latory signalling pathway initiated by physioiogicdl Schiff-base

formation. The leading candidates for physiological mediation of

Schiff-base formation are described below.

Mechanism of action of Schiff-base-forming drugs How does the formation of a Schiff base by small exogenous mol-

ecules on specialized T-cell-surface amines provide a costimulatory

signal to the T cell? Electron-probe, X-ray-microanalysis studies re-

vealed that the formation of a Schiff base on T-cell-surface amines

produces large changes in the levels of intracellular potassium (K+)

and sodium (Na+)” (Fig. 4). The resting state of h$h K+, low Na+ is

reversed by tucaresol within 5 min. By 2 t, levels have reverted to

the resting state. These effects are prevented by ligation of cell-

surface amines with S-NHS-biotin and also by the K+ channel

antagonist, clofilium tosylate. Clofilium likewise inhibits the co-

stimulatory effects of tucaresol in terms of enhancement of IL-2

production by T cells. Convergence of tucaresol costimulation with

the TCR-dependent pathway has been identified at the level of the

mitogen-activated protein kin? .r (MAPK), ERK2 (H. Chen, M.

Rogers and J. Rhodes, unpublis’.; d).

MAPK is a family of serine/thr ,?onine kinases activated by tyrosyl

phosphorylation and is important in the dow&ream integration

IMMUNOLOGY TODAY

and transmission of a wide range of mito-

genlc and activational extracellular sigzlals24.

Studies in the Jurkat J6 T-cell line show that

stimulation with tucaresol alone, or T-cell re-

ceptor (TCRI-directed stimulation with anti-

CD3 (in the absence of costimulation), induces

tyrosyl phasphorylation of MAPK. When

both stimuli are given together, MAPK

phosphorylation is substantially enhanced

and prolonged. A selective synthetic inhibitor

of MAPK tyrosyl phosphorylation, (2-[2’-

amino3’-methoxyphenyll-oxanaphthalen-4-

one), which acts at the level of the MEK ki-

nase one step upstream of MAPK (Ref. 25),

prevents both the phosphorylation induced

by tucaresol and the costimulatory effects of

tucaresol in terms of enhancement of IL-2 pro-

duction in response to TCR/CD3 stimulation.

While some of the important even% in

the costimulatory pathway exploited by

tucaresol are being characterized, other

equally important questions remain unan-

swered. Amongst these are the identity of

the celi-surface macromolecules that trans-

duce a costimulatory signal in response to

Schiff-base formation on functionally sensi-

tive amino groups. What kind of macromol-

ecular process do we envisage taking place?

-~~ ----------- __-__ - d a- \ , COOH

Fig. 4. The formation of a Schiff base by fztcaresol 011 specialized T-cell-surface amines provides a

costimulatory signal to the T cell through a nzechanisnz fiznt activates sodium and potassium trans-

porf. Tucaresol mediated costinzulation cozzverges zoith TCR sigzzalling at the level of tyrosyl phos-

plzoryiation of the MAX, ERK’. Abbreviations: AK, antigerz-presenting cell; MAPK, nzitogerz-

activated profeizz kinase; MIX, nzajor lzisfocompatibility conzplex; TCR, T-cell receptor; T/z, T lzelper.

The physiological precedents (see Box. I) suggest a mechanism in

which Schiff-base formation takes place on conformationally piv-

otal lysyl e-amino groups expressed in the ligand-binding site of in-

tegral membrane proteins. Such an event could induce a conforma-

tional change in the macromolecule thereby transmitting a signal

across the plasma membrane.

One of the initial observations on Schiff-base formation in the in-

duction of immune responses showed that erythrocyte (E&rosette

formation at room temperature provided an analogue for Sctuff-

base formation in APC-T-cell inductive interactionsz6. E-rosette for-

mation reflects macromolecular interactions between CD2 and its

ligands, especially leukocyte function-associated molecule 3 (LEA-3).

These observations together with the markedly similar profile of

CD2 (Ref. 26) and tucaresolzz costimulation, make CD2 an attractive

candidate as a principal receptor for S&&base costimulation. The

recently described three-dimensional structure of glycosylated human

CD2 reveals certain structural features that we might expect of a re-

ceptor for Schiff-base costimulation in the form of an unusual con-

centration of surface-exposed lysine rcsiclues in the LFA3-binding

domainz7. The e-amino groups of these lysines are conformationally

pivotal, contributing a destabilizing concentration of positive charge

that, in the absence of an N-linked glycan opposite the binding site,

unfolds the polypeptide. The N-linked glycan appears to function

by stabilizing the polypeptide in the folded conformation through

hydrogen bonds and van der Waals contacts, producing a dynamic

rather than a static stability. These features of conformhationalk7 dy-

namic c-amino groups in the binding domain of CD2 are those ex-

pected of a target for Schiff-base signalling across the plasma mem-

brane. We know from direct labelling and immunoprecipitation

studies that tucaresol reacts with CD2, and a number of techniques

are currently being used to assess whether tucaresol initiates a sig-

nal through CD2. However, only direct evidence will be persuasive

and it should be noted that some evidence does not support an es-

sential role for either the carbohydrate elements or conformational

change in ligand=bmding or signal transmission by CD2 (Refs 28,29).

Another intriguing question is the nature of the physiological

donor of carbonyl groups that is mimicked by costimulatory Schiff-

base-forming drugs. As discussed previously, in all cases where

Schiff-base formation is important in a physiological process, the

carbonyl donor is an organic prosthetic group (e.g. pyridoxal phos-

phate, pyrroloquinoline quinone, retinaldehyde). Vitamin A ap-

pears to play an essential role in the immune system (see Ref. 30),

and work is needed to identify a specific locus of action. Similarly,

in APC-T-cell interactions it is vital to identify a physiological donor

of Schiff-base-forming carbonyl groups, a function in which retin-

aldehyde, in other physiological systems, is a principal player. Of

course there may be several loci of action for vitamin A in immun-

ity, and one that does not appear to involve retinaldehyde is in the

process of being elucidated3z. Pyridoxal phosphate is also an attrac-

tive candidate in view of increasing evidence suggesting an essen-

tial role for its precursor, vitamin B6, in T-cell-dependent immune

responses . ‘* A number of techniques are currently being employed

SEPTEMBER I996

Organic prosthetic groups: Pyridoxal prosphate? Retinal?

4 0 TCR

Na+

Na+

GTP GDP

Phosphorylation of MAPK

ERK2

Fig. 5. Possible macromolecular mecktrisms i:r Sclriffbase cosfimulutiorr. (1) CD2 is RH ntfmcfiv~ candidate as a prirlcipal receptor for Schiffbase cosfimulnfion. (2) Many physiological precedents

suggest that fhe natural donor oJSchiff-base-forming carbonyls is likely fo bc an organic prosfhefic

group such as retinal or pyridoxal phosphafe. (3) and (4) The early evetlfs linking signal fransdtrc- fion to ion transport and fyrosyl phosphorylafion have yet to be characterized. Abbreviations:

MAPK, mifogen-acfivnted protein kinuse; TCR, T-cell recepfor.

to address these questions. The light-sensitivity of organic proa- thetic groups could potentially explain some of the photolabile fea- tures of the immune response in terms of immunosuppression and anergy induced by W light treatment in viva and in uifroU,34 (see Fig. 5).

Significance of the Schiff-base mechanism in immunopathology The impcrtance of the Schiff-base mechanism in immune activation illuminates an entirely new area in immunopathology and there are a number of intriguing possibilities that are being actively investi- gated. For example, (1) bacterial abscess formation induced by amines on capsular polysaccharide (e.g. of Bacferuides fiagilis) might be due to aberrant si~nalling initiated by the reaction of these groups with functionally important cell-surface carbonyls of lymph* cytes 35,X; (2) Schiff-base-forming ketones and aldehydes are gener- ated in the inflamed synovium in rheumatoid arthritis and might exert immunomodulatory effects that contribute to the disease process 37; (3) the Schiff-base mechanism also provides a direct mechanism for the induction of immune damage in alcoholic hepatitis38 - acetaldehyde, the primary metabolite of ethanol, is a potent immunopotentiator ilz vitro and in vmo by virtue of its Schiif-base-forming reactivity”; (4) the studies of Page and colleagues on outbreaks of serious, food-related autoimmunotoxicity

have shown reactive species, such as the Schiff-base, l,l’-ethylidenebis Wryptophan) (EBT), decompose to a very reactive carbino- lamine, raising the possibility that there is a direct effect on lymphoid receptors sensitive to Schiff-base-forming molecules involved in

autoimmunotoxicity3g.

An oraiiy active, mechanism-based drug

that acts systemically to potentiate the im- mune system, and biases immunity towards the cell-mediated Thl-type response, should

have potential applications in a wide range

of chronic, infectious diseases caused by

intracellular pathogens. These would include: viral infections such C-IS chronic viral hepa-

titis and human immunodeficiency virus

(HIV); mycobacterial infections such as tu-

berculosis and leprosy; protozoa1 parasitic

infections such as malaria and leishmania-

sis; as well as other parasitic diseases where

cell-mediated immunity is likely to be

important. As an immunopotentiator, tu-

caresol is likely to be complementary to vac-

cine therapy in which the aim is to provoke

cell-mediated or Thl-type immunity to pathogenic antigens. Such

vaccine-therapy strategies are currently being developed for the

treatment of chronic hepatitis B virus infection, HIV and malignant

melanoma, using a variety of subunit immunogens designed to

stimu!ate Th and cytotoxic T-cell responses in association with suit-

able local physicochemical adjuvants4Mz. Oral administration of

tucaresol during vaccine therapy would provide a unique means of

strengthening the desired immune response and this would not

preclude the use of local adjuvants (unlike tucaresol, adjuvants, by

their nature, cannot be used systemically). Alternatively tucaresol

could potentially be used locally as an adjuvant with or without

additional oral administration. Finally, the Schiff-base mechanism

might provide an explanation for the potent adjuvant effects of

Quillaja saponins such as QS21 whose effects are dependent on a re-

active (Schiff-base-forming) aldehyde43, as well as the success of the

adjuvant strategy employing mucins conjugated with oxidized

faldehyde-bearing) mannan to select for a Thl-type response”.

Concluding remarks Costimulatory Schiff-base-forming drugs, as exemplified by tucaresol, provide the first mechanism-based, orally active immuno- potentiatory therapy for clinical testing. The fact that this strategy is

entirely new means that it is untested against disease targets.

However, pilot clinical studies have been initiated in chronic hepatitis

IMMUNOLOGY TODAY

B virus infection, HIV infection, and in malignant melanoma.

Convincing evidence of clinical efficacy can only come from the re-

sults of carefully controlled clinical trials in potential disease indi-

cations, where disease progression and parameters cif immune re-

sponsiveness can both readily be measured and quantitated. For

example, responses in phase I/II clinical trials will be measured

both in terms of disease parameters and T-cell responsiveness bn

disease specific and nonspecific antigen, anti-CD3 and mitogen.

Readouts will in&de proliferative responses and production of Thl-

and Th2-type cytokines. The aim of immunopotentiatory therapy is

to tip the balance of the contest between chronic infection and

immunity in favour of the immune system, with the minimum ef-

fective dose of the drug. With the proper assessment of risk vs. ben-

efit and appropriate choice of therapeutic regimen, costimulatory

Schiff-base-forming drugs provide a new opportunity for testing an

orally active, mechanism-based immunopotenliatory therapy in

chronic, infectious diseases and cancer.

John Rhodes is at the hmunoiogy Unit, Division of Cell&r Sciences,

Glaxo Wellcome Medicines Research Centre, Gunnels Wood Road,

Stevenage, Hertfordshire, UK SGI 2Ny.

23 Clerici, M. and Shearer, G.31. (1994) /n~r~ur~~ol. ~odny 15, 575-581

24 Seger, R. and Krebs, E.G. (1995) FASER. 1, 9, 720-35

25 hdky, D.T., Pang, L., Decker, S.J., Bridges, A 1. and Saltiel, A.R. (1995)

Proc. N&f. Acnd. Sci. U. S. .4. 92,7686-7689

26 Hargreaves, R., Logiou, V. and Lechler, R. (1995) Iuf. hnn~unol. 7,

1501-1513

27 Wyss, D.F., Choi, IS., I.!, ;_ c! .i:. (lS%i Science 269, 1273-1278

28 Bodian. D.L., Jones, E.Y., Harlos, K. et nl. (1994) Stnrcfure 2, 7S5-766

29 Davis, S.J., Davis, E.A., Barclay, A.N. et rd. (1995) 1. Biol. Chem. 270,

369-375

30 Malkovsky, M., Medawar, I?B., Thatcher, D.R. rf of. (1985) Pmt. Nflh.

Acad. Sci. U. S A. 82,536-538

31 Derguinin, F., Nakanishi, K., HBmmerling, U. and Buck, J. (1994)

Biochcmisfty 33, 623-628

32 Rail, L.C. and Meydani, S.N. (1993) Nufr. Rev 51, 217-225

33 Kripke, M.L. (1994) Cower RI-L 54 6102-6105

34 Ulbich, S.E. (1995) 1. Itlocsf. Dermorol 105 (Suppl. I), 305-365

35 Tzianabos, A.O., Onderdnnk, A.B., Kosner, B., Cisnertos, R.L. and

Kasper, D.L. (1993) Srhcc 262. 416-4lY

36 Tzixabos, A.O., Onderdonk, A.B., Zaleznik, D.F., Smith, R.S. and

Kaspar, D.L. (1994) Infect. Imrmm 62, 3590-3593

37 Edmonds, S.E., Ellis, G., Gaffney, K., Archer, 1. and Blake, D.R. (19Y5)

Scn~id. j. Rhenmnfol. (Suppl.), 101, 163-168

38 Sherman, D.1. and Williams, R. (1994) Br. Med. Bull. 50, 124-138

39 Brenncman, D.E., Page, S.W., Schultzberg, M. ef al. (1993) /. Plmml. Exp.

Then 266, 1029-1035

4;! :m ’ c ‘1995:. Biarncd P’-,-,-:!xr .;‘I::v. 49, 10L%109 .,., -. . .

41 Redfield, R.R. and Birx, D.L. (19Y2) AIDS Rr. HIW. R&ozlirrrses 8,

1051..1058

42 Parth, A,, Hoon, D.S., Foshag, L.J. IQ nl. (1994) Corur Rcs. 54, 3342-3345

43 Soltysik. S., Wu, J-Y., Reccbia, J. et nl. (1995) Vncci?lc 13,1403-1410

44 Apostolopoulos. V., Pietersz, GA., Loveland, B.E. rt nl. (1995) Pror.

Nnfl. Aaid. Sci. U. S. /1. 92, 10128-10132

References

1 Novogrodsky, A. and Katchalski, E. (1972) Proc. NaH. Acnci. Sci. U;S. A.

69,3207-3210

2 Novogrodsky, A. and Katchalski, E. (1973) Proc. Nafl. Acad. Sci. U. S. A.

70,1824-1827

3 Greineder, D. and Rosenthal, A. (1975) J. Innmrnol. 932-938

4 Rosenthal, A., Blake, J.T., Ellner, J.J., Greineder, D.K. and Lipsky, l?E.

(1976) in bnmunobiology of fhe Macroplmge (Nelson, D.S., ed.), pp. 131-160,

Academic Press

5 Rhodes, 1. and Tite, j. (1988) J. bnmrozol. 140,3344-3351

6 Snell, E.E. and Dimari, S.J. (1970) in The Enzynres: Kinetics nrrd Meclmrzisn~ Nol. 2) ( Boyer, P.D., ed.), pp. 335-346, Academic Press

7 Fherst, A. (1984) in Enzyme Structure and Meclznnism (21v.l edn), pp. 69-97,

W.H. Freeman

8 John, R.A. (1995) Biochint. Biophys. Actn 1248,81-96

9 van der Meer, R.A. aad Duiie, J.A. (1986) Biochenf. I. 239,789-791 10 Shyer, L. (1988) in Bioche,nistry (3rd edn), pp. 1005-1041, W.H. Freeman

11 Rhodes, J., Chen, H., Hall, S.R. et a[. (1995) Nnfure 377, 71-75 12 Mukchata, Y. (1994) Biophys. Chenl. 50,191-201

13 Sasaki, J., Brown, L.S., Chon, Y-S. et al. (1995) Science 269, 73-75

14 Rhodes, J. (1989) 1. bnmunol. 143, 1482-1489

15 Gao, X-M. and Rhodes, J. (1990) 1. Immurrol. 144,2883-2890

16 Rhodes, J. (1990) J. Immt~nol. 145,46H69

17 Rhodes, J., Zheng, 8. and Lifely, M.R. (1992) Intm~mology 75,629431

18 Zheng, B., Brett, S., lite, J.l?, Lifely, M.R., Brodie, T.A. and Rhodes, J.

(1992) Science 256,1560-1563

19 Rhodes, J., Zheng, B. and Morrison, CA. (1995) in Combined Voccirtes and

Simultnneous Administmtions: Current Issues and Perspectives (Williams, J.C.,

Goldenthal, K.L., Burns, D. and Lewis, B.P., Jr, eds), Ann. N~UJ York Acnd. Sci.

754,169-186

20 Rolan, l’.E., Pa:ker, J.E., Gray, S.J. et al. (1993) Br. J. Plwmnc~l. 35,

419-425

21 Seder, R.A. and Paul, W.E. (1994) Annu. Rev. Immunol. 12,635-673

22 Del Prete, G., Maggi, E., Romagnani, S. ef al. (1994) Lnb. hmesf. 70,299-306

The Immunology Today W Envirtmmsnt,

including immunology Today Online

Have a look at our homepage at the following URL

httpd/www.elsevier.nUbcate/ito

This Web sita will act as a ‘rocus for providing access to the we&h of articles in Immunology Today On&n and will be updated on a monthly basis to keep you informed of current and upcoming articles in /rnmin&gy TO@ and other +&s journals. Them are also stepping stones to other Web sites of immunological interest

lf yw know of a useful resource that we should be aware of, why not let us know &

IT@etder.co.uk