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References

1 Ishizaka, K. (1976)Adv. ImmunoL 23, 1-75 2 Levine, B. B. and Vaz, N. M. (1970)Int. Arch. Allergy. Appl. Irnmunol. 39,

156-171 3 Katz, D. H. (1980) Immunology 41, 1-24 4 Jarrett, E. E. E. and Stewart, D. C. (1972) Immuno/ogy 23, 749-755 5 Ishizaka, K., Seumura, M., Yodoi, J. and Hirashima, M. (1981) Fed.

Proc. Fed. Am. Soc. Eap. BioL 40, 2162-2166 6 Gonzalez~Molina, A. and Spiegel, H. L. (1977)J. Clin. Invest. 59,

616-624 7 Fritsche, R. and Spiegelberg, H. L. (1978)J. Immunol. 121,471-478 8 Yodoi, J. and Ishizaka, K. (1980)J. Immunol. 124, 1322-1329 9 Yodoi, J , Hirashima, M. and Ishizaka, K. (1981)J. Immunol. 126,

877-882 10 Spiegelberg, H. L., O'Connor, R. D., Simon, R. A. and Mathison,

D. A. (1979)J. Clin. Invest. 64, 714-720 11 Yodoi, J,, Hirashama, M. and Ishizaka, K. (1981) J. ImmunoL 127,

471-475 12 Hirata, F., Sehiffmann, E., Verkatasurbramanian, K., Solomon, D. and

Axelrod, J. (1980)Proc. NatlAcad. Sa'. USA 77, 2533-2536

Immunology Today, vol. 4, No. 7, 1983

13 Yodoi, J., Hirashima, M., Hirata, F., DeBlas, A. L. and Ishizaka, K. (1981)J. ImmunoL 127,476-482

14 Hirashima, M., Yodoi, J. and Ishizaka, K. (1981)J. Immunol. 126, 838-842

15 Hirashima, M., Yodoi, J. and Ishizaka, K. (1980)J. lmmunol. 125, 2154-2160

16 Hirashima, M., Yodoi, J., Huff, T. and Ishizaka, K. (1981)J. Immunol. 127, 1810-1827

17 Hirashima, M., Yodoi, J. and Ishizaka, K. (1981)J. Immunol. 127, 1804-1809

18 Yodoi, J., Hirashima, M and Ishizaka, K. (1981)d~ Immunol. 127, 1579-1585

19 Chen, S. S., Bohn, J. W., Liu, F. T. and Katz, D. H. (1981) J. ImmunoL 127, 166-173

20 Kishimoto, T., Hirai, Y., Suemura, M. and Yamamura, Y. (1976)J. Imraunol. 117, 396--404

21 Suemura, M., Kishimoto, T., Hirai, Y. and Yamamura, Y. (1977)J. lmmunoL 119, 149-155

22 Kishimoto, T., Hirai, Y., Suemura, M., Nakanishi, K. and Yamamura, Y. (1978)J. Imnmnol. 121, 2106-2112

23 Hirashima, M., Uede, T,, Huff, T. and Ishizaka, K. (1982)J. Immunol. 128, 1909-1916

24 Ishizaka, K. and Sandberg, K. (1981)J. Immunol. 126, 1692-1696

The single-cell assay in cell-mediated cytotoxicity Benjamin Bonavida, Thomas P. Bradley and Elizabeth A. Grimm

Studies on the humoral antibody response were greatly advanced by the introduction in 1963 of the Jerne plaque assay which permitted direct enumeration of individual antibody-producing plasma cel~. Studies of cellular cytotoxicity were limited until recently to the examination of whole populations of oCfector cells 2. Here, Benjamin Bonavida and his

colleagues describe an assay in agarose which allows the study of single cytotoxic cellf1-5.

Cytotoxic T lymphocytes (CTL) must bind to target cells before lysis occurs; cytotoxic cells thus adhere to target- cell monolayers n. Our first attempt to demonstrate killing at the single-cell level took advantage of this observation. When C T L were dispersed on the surface of monolayers and incubated, zones or plaques oflysis in the monolayers were obtained. This method provided only a minimal estimate of the frequency of killers since plaque formation required an effector cell to kill four or more targets in a short-term assay 7. Berke et aL 8 and Martz ° demonstrated the binding of effector to target cells in suspension. Lymphocyte-target-cell interactions were studied in liquid medium in a hemocytometer ~° or in microwells after micromanipulation", and lysis was determined by the loss of target-cell optical refractiveness.

The limitations of these techniques gave rise to the idea of immobilizing the effector-target conjugates which were a prerequisite for target killing, by diluting them in agarose and then incubating them to permit target-cell lysis. After staining, the dead target cells are counted microscopically. Each stained (dead) conjugate target cell indicates that the adjacent lymphocyte was indeed a cyto- lytic cell. Thus, the frequency of killer cells can be easily estimated t2,'3.

The single-cell assay complements previous indirect methods used in measuring cytotoxicity (for review see

Benjamin Bonavida and Thomas P. Bradley are in the Depart- ment of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA 90024, USA; Elizabeth A. Grimm is in the Surgery Branch, National Cancer Instittxte, National Institutes of Health, Bethesda, MD 20205, USA.

Ref. 2) by permitting direct examination of the cellular characterisitcs and lytic mechanisms of single effector cells. It was first used to study alloimmune cytotoxic T lymphocytes (CTL) 3 but immediately applied by us and others to other cytotoxic ceils, including natural killer cells (NK) 14'16, antibody-dependent killer cells (K cells in ADCC) 17,1s, and cytotoxic macrophages 19. The assay is applicable in many species such as man, mice, rats, frogs, rabbits and lampreys. It is discussed in detail in references indicated in Table I and applications of the single-cell assay are outlined in Table II.

Enumeration and characterization of cytotoxic cells Frequency of effector cells which bind to targets

All single-cell assays rely on the requirement ofeffector cells to interact with and bind to target cells. Centrifuga- tion facilitates binding. The frequencies of effector cells which bind to target cells in several systems are summar- ized in Table III. In most systems there is some non- specific binding. Neither its mechanism nor functional relevance are known but in most circumstances binding seems to be related to recognition by the effector cell, and thus a high frequency of specific binding above back- ground level is observed.

The recognition event in CTL-mediated lysis is antigen-specific, suggesting that binding is the result of interaction between antigen receptors of T cells and tar- get antigens. Binding in other effectors probably involves different receptors. For instance, mouse CTL recognize antigens primarily encoded in the K and D regions of the MHC but mouse NK cells recognize an entity common to

©1983, Elsevier Science Publishers B.V., Amsterdam 0167- 4919/03/$01.00

Immunolog7 Today, vol. 4, No. 7, 1983

TABLE I. Single-cell assays used in binding and cytotoxicity

Method System References

Clusters of CTL-bound targets in suspension CTL

Conjugates of CTL-bound targets in suspension CTL

Cytolytic plaque assay CTL Conjugates in agarose

Single target-single effector

Double target-single effector

Single target-multiple effector

8,10,11 7

CTL 2-5, 13, 26 LDCC, ODCC 5, 13, 28 NK 13-16, 18, 28 K 13, 16, 17 M~ 19 CTL 5, 13 LDCC, ODCC 5, 13, 28 NK 13, 28, 29 ADCC 13, 29 CTL E. A Grimm

eta/., unpubl. obs.

TCGF: T-cell growth factor; ADCC: antibody-dependent cellular cytotoxicity; LDCC: lectin-dependent cellular cytotoxicity [in which lectins (concanavalin A or phytohemagglutinin) induce non-specific cytolysis of syngeneic or xenogeneic targets by alloimmune lympho- cytes (mouse) or normal blood lymphocytes (man)I; ODCC: oxidative- dependent cytotoxicity in which targets are modified by periodate or neuraminidase and galactose oxidase.

all N K targets and distinct from the K- and D-encoded structures 2°. A D C C requires an interaction between a receptor on the effector cell and the Fc region of antibody- coated target cells 21. The molecular na ture of these various receptors and how they signal the effector cell for lysis is unknown but b ind ing is a requirement for lysis. Since the b inding frequency can be altered by various factors, the frequency of binders in a cytotoxic system against a particular target may have biological and clinical significance. Thus , a correlation may be made between the frequency of binders and the frequency of killers unde r specific conditions.

Frequency of cytolytic effector cells which lyse bound target cells First attempts to determine the frequency of killer cells

within a cell populat ion using a 51Cr-release assay and describing lysis in terms of enzyme-snbst ra te inter- actions 22, were inconsistent and required m a n y assump- tions, main ly that a single effector cell can interact and bind with only one target cell and that the rate of lysis by individual ceils is constant 23. From the single-cell assay, by direct measurement , we know that effector cells can recycle and that ceils are inherently heterogeneous in their lytic efficiency 3'4. Target-ceil death is detected either by dye exclusion s or by the loss of optical refractiveness '1. These methods corrrespond well to other measures of cell death, such as alteration in cell permeability, detachment from monolayers, failure of division and release of radio- activity2L

SuoCace phenotype of individual ~fector cells The surface phenotype of the effector or target cells in a

conjugate can be easily discerned by indirect i rnmuno- fluorescence. In this way mouse cytotoxic T lymphocytes bound simultaneously to both specific and non-specific targets were found to be Lyt 2 ÷ (Ref. 5). Other probes to

197

define the effector cell surface phenotype include ferritin- labeled antibodies directed against surface components of choice 25. Electron microscopy has also been used to view unique membrane configurations that might reveal ultrastructured aspects of the lytic reaction 2s.

Preselection of conjugates for analysis An adaptat ion of the single-cell assay in agarose

involves the uses ofa microlocater grid to select a particu- lar conjugate for viewing throughout the various lytic post-binding stage¢. After being immobilized but before incubation, the location of a conjugate on a grid matrix is recorded, so that the conjugate can be relocated in order to view the lytic process. Relocation is also an advantage when studying effector cells combined with two morpho- logically similar types of target cell (one being stained with fluorescein dye before incubation) and effector cells morphologically indistinguishable from target cells, the latter being fluorescein-dyed. After incubation, the con- jugates of interest are relocated and the identity of lysed targets assessed by t rypan blue dye exclusion. It may also be possible to mechanically isolate prelocated killer cells for further study.

Killing of more than one type of target by a single effector cell Is a single effector cell restricted in its specificity to

killing only a single type of target cell? This question can be addressed with the single-cell assay. Zagury et aL have shown that multiple targets bound to a single effector cell are killed at different times sequentially2L In addition, a single C T L kills two simultaneously bound non-identical targets when one target displays the sensitizing antigen and the other target is syngeneic to the effector (i.e. not recognized by it) and coated with lectin 5. This two-target conjugate technique (described above) can also be used to delineate subpopulations within the immune system. Thus, in man, cytotoxic cells able to kill NK-sensit ive targets were shown to be distinct from the cells that kill lectin-coated targets (LDCC) 2s, while some N K cells could kill antibody-coated, NK-resis tant targets 29.

Competition for binding Specificity in cytotoxicity, when assessed by testing the

lytic potential of an effector populat ion against a battery of target cells, does not establish whether the effectors are a mixed or single population. Landazur i and Herber- m a n ~° have developed a cold-target cell competit ion assay us ing 51Cr release in which unlabeled target cells are added to a constant n u m b e r of labeled target cells and

TABLE II. Applications of the single-cell cytotoxicity technique

1. Establishment of frequency of cells which bind to target ceils 2. Establishment of frequency of the cytotoxic cells among binders 3. Determination of surface phenotype of the effector or target cells

by immunofluorescence 4. Determination of the effector-cell and target-cell type specificities

using the two-target conjugate assay 5. Investigation of the mechanism of lysis at distinct stages in the

lytic process 6. Determination of effector-cell cytotoxic efficiency and activation 7. Ability ofcytotoxic cells to recycle 8. Application in clinically related studies

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TABLE IIL Frequency of binders and killers

Immunology Today, vol. 4, No. 7, 1983

Cytotoxic cell Effector populations Species % Conjugates b % Killers c References

T lymphocyte Allosensitized peritoneal lymphocytes (in vivo) Mouse 20-50 20-50 3, 5 Allosensitized spleen cells (in vivo) Mouse 15-25 7-13 3 Allosensitized spleen cells (in vitro) Mouse 3-10 3-17 3 Lines cloned using TCGF a Mouse 10-20 10-25 Nylon-wool-purified spleen cells Mouse 20-40 20-30 3 Peripheral-blood lymphocytes Man 8-15 3 14, 28, 29 Large granular lymphocytes Man 30-50 20-40 28, 39 Large granular lymphocytes Rat 40

ADCC a effector Peripheral-blood lymphocytes Man 8-15 2-3 17, 29 Large granular lymphocytes Man 30-50 20-40 29, 39

LDCC a and AUosensitized peritoneal lymphocytes (in vivo) Mouse 20-50 20-50 5, 13 ODCC effector

Peripheral-blood lymphocytes Man 20-35 8-15 13, 28

• For abbreviations see Table I. b % Effectors which bind to targets. c % Target-bound effectors which kill attached target.

cytotoxicity is measured. I f the non-labeled target cells are antigenically related, they compete with the labeled target cells and there is less killing. In an analogous system using single cells, Berke et al. 31 have reported competi t ion at the level of conjugate formation. The target cell is labeled

Fig. 1. Two NK conjugates. The conjugates (consisting of human peripheral-blood lymphocytes

bound to the NK-sensitive targets K562) have been immobilized in agarose, plated onto a glass slide and used in the single-ceU assay. After an incubation for three hours at 37 °C, the slide is stained in trypan blue and fixed in formaldehyde. The upper conjugate is an example of a target which has been lysed by the attached lymphocyte (note that the target has been stained blue and has lost refractivity). The lower conjugate consists of a viable target which has not been lysed by the bound lymphocyte (note the high degree of refractivity of the viable target).

with fluorescein and the inhibit ing targets are unlabeled. If there is competi t ion then fluorescent conjugates are fewer. The frequency of both antigenically specific and non-specific cells can be examined, as well as the specificity of target antigens.

Cell enrichment C T L can be enriched by first b inding them to allo-

geneic target cells and layering the suspension on a dis- continuous fetal calf serum gradient. Conjugated lym- phocytes separate from free targets because of the differ- ence in size 12 and the bot tom layer contains up to 90% conjugated lymphocytes, an average of 70% of these lysing their attached target. The lymphocytes bound to targets can be dissociated and separated from the target cells, thus enabling enr ichment ofcytotoxic cells.

Sensitivity of assay In conventional whole-populat ion assays for cytotoxi-

city a large number of cytotoxic cells must be present in the effector populat ion for reasonable chance of detecting cytotoxicity. In the single-cell assay, however, it is theoretically possible to detect a single killer cell f rom a mixed populat ion of cells, and so a much smaller number of cells (< 1 000) is needed 12.

Studies on the m e c h a n i s m of ce l l -med ia t ed cy to tox ic i ty Evaluation o/distinct stages of the lyric process

Although the mechanism under lying cell-mediated cytotoxicity is not known, it is proposed that various stages lead to the cytolytic event. Mar t z has shown that binding, p rogramming for lysis, and a lethal hit stage are distinct ~2. Agents which block binding can be uniquely evaluated with the single-cell assay. W e have investigated the blocking of cytolysis by ant ibody as a probe to de- lineate the steps involved in lysis. For example, anti-Lyt 2 antibody and R A T * (rat an t i -mur ine activated T cells) antibody block cell-mediated cytotoxicity in the absence of complement . The single-cell assay showed that anti- Lyt 2 antibodies block effector-target interaction 33 while R A T * blocks cytotoxicity post-binding0~.35. These anti- sera presumably recognize different molecules on the ef-

Immunology Today, vol. 4, No. 7, 1983

lector cells, each of which may be involved in the mechan- ism o f ly s i~ .

Kinetics of lysis After b ind ing to a target, some C T L take longer than

others to kill itL The rate of killing by cytotoxic cells is not constant and cells at different states of differentiation or matura t ion kill at different rates. For example, single-cell techniques have shown that C T L generated in a second- ary response are faster killers than their counterparts in the pr imary response3'L

The single-cell assay allows examinat ion of agents which enhance cytotoxicity. In the N K system t reatment of the effector cells by interferon did not alter the n u m b e r of b ind ing cells but raised the frequency of killer cells and enhanced the kinetics of lysis, suggesting that the rate of killing was increased and that some cells which b ind and do not kill (non-lytic, p re -NK cells) can be triggered by interferon to kill.

L ysis of single targets bound by multiple effectors In cultures containing mouse C T L , mult iply bound

targets can be seen if the effector/target cell ratio is increased but the total n u m b e r of b ind ing lymphocytes is lower than the n u m b e r observed at the s tandard 1 / 1 ratio, indicating that b ind ing avidity is heterogeneous.

W h e n mult iply b o u n d targets were incubated in the single-cell assay, and the kinetics of lysis followed, we found that targets b o u n d by one, two or three lympho- cytes lysed at rates of 1.3 _ 0.1, 1.9 _ 0.2 and 3.3 _ 0.4 per hour, respectively. In all cases, the lysis followed first- order enzyme kinetics, sugesting that when simultan- eously bound to the target, the effector cells operated independendy, with the fastest killer inducing lysis. These data suggest an association between b ind ing avidity and lytic efficiency (E. A. Gr imm, L. Roos and B. Bonavida, unpublished observations).

Recycling of effector cells to kill more than a single target The single-cell technique has established that a cell

which has killed one target can recycle to kill others. Zagury et al. showed by micromanipulat ion that single effector cells can kill a n u m b e r of targets i n sequence 11, and Mar tz 32 earlier detected recycling in a mixed populat ion of effector ceils when targets were added in sequence. The single-cell assay analyses the recycling of individual cells within the mixed effector-cell population. The fact that physical movement and hence recycling is prevented in the agarose single-cell assay explains why killing in this assay sometimes does not correlate with killing in 51Cr-release assays where recycling occurs.

Nature of the lytic lesion and the delivery of the lethal hit Time-lapse cinematography of C T L - t a r g e t cell con-

jugates dur ing the single-cell assay and observations of the entire lytic process at the light-microscope level 37 showed that the b ind ing of effector to target cells was the initial event, bu t cont inued at tachment of the lymphocyte to the target was not necessary for lysis to progress. This initial a t tachment corresponds directly to the steps termed ' p rogramming for lysis' by Mar tz ~2. The stage of target-

199

cell death varied in time, from 15 minutes to as long as several hours. Dur ing death, the target cell undergoes a un ique process of swelling and stretching, ( 'zeosis ') which differs in appearance from the killing induced by ant ibody and complement.

By electron microscopy a n u m b e r of poorly defined junction-l ike structures br idging the connection between C T L and target cell were observed 26. Such structures might provide a conduit for transfer of lytic molecules or they could be responsible for the very strong b inding necessary to damage a target cell by a shearing force. No obvious secretory apparatus was visible at the junc t ion region.

Clinical applications The immune system is adversely affected in m a n y

diseases and in patients treated with immunosuppressive agents. Convent ional cytotoxicity assays cannot indicate the level of defect bu t single-cell assays can determine if the defect is due to: (a) an absence of cells that can recognize and b ind targets; (b) preservation of b ind ing but failure oflytic mechanisms; (c) decrease in frequency of killer cells; (d) slowed kinetics of lysis; (e) loss of 'recyclability' . As an example, N K lytic function in both Chediak-Higashi disease 37 in m a n and the beige mu tan t in mice 3s is deficient, not because b inding is absent but because the N K cells lack the ability to lyse the bound targets.

Changes in immunological status after t reatment and studies of ontogeny and phylogeny can also be evaluated with the single-cell assay•

Conclus ion The single-cell assay is a valuable means of s tudying

different types of cell-mediated cytotoxicity directly and allows examinat ion of m a n y aspects of effector function and target specificity. In the future, it should be possible to devise computerized ins t rumenta t ion able to visualize, record, and count b inder and killer cells with precision, rapidity and reproducibility.

Acknowledgements Work in this laboratory has been supported by Grants CA12800,

CA19753, and CA24314 from the National Cancer Institute, USPHS.

References 1 Jerne, N. K. and Nordin, A. A. (1963) Science, 140, 405-406 2 Bonavida, B. and Bradley, T. P. (1980) Immunol. Today 5, 104-109 3 Grimm, E. A. and Bonavida, B. (1979)J. Immunol. 123, 2861-2869 4 Grimm, E. A. and Bonavida, B. (1979)J. Immunol. 123, 2870-2877 5 Bradley, T. P. and Bonavida, B. (1981)J. Immunol. 126, 208-213 6 Berke, G. and Levey, R. (1972)J. Exp. Med. 135, 972-984 7 Bonavida, B., Ikejeri, B. and Kedar, E. (1974) Nature (London) 249,

658-659 8 Be~:ke, G., Gabison, D. and Feldman, M. (1975) Eur. J. Immunol. 5,

813-818 9 Martz, E. (1975)J. Immunol. 115, 261-267

10 Zagury, D., Bernard, J., Thiemess, N., Feldman, M. and Berke, G. (1975) Eur. J. Immunol. 5, 818--822

11 Zagury, D., Bernard, J., Jeannesson, p., Thierness, N. and Duffer, J. (1975) Ann. Immunol. (Paris) 126C, 23

12 Grimm, E. A. and Bonavida, B. (1977)J. lmmunol. 119, 1041-1047 13 Bonavida, B., Bradley, T. P. and Grimm, E. A. Methods Enzymol. (in

press) 14 Silva, A., Bonavida, B. and Targan S. (1980)J. Immunol. 125, 479-484 15 Jondal, M. and Merril, J. E. (1981)Eur. J. Immunol. 11,531-535 16 Merril, J. E., Ulberg, M. and Jondal, M. (1981)Eur. J. lmmunol. 11,

535-541

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17 Neville, M., Grimm, E. A. and Bonavida, B. (1980)J. lmmunoL Method. 36, 255-268

18 Targan, S., Grimm, E. A. and Bonavida, B. (1980)J. Clin. Lab. Immunal. 4, 165-168

19 Fischer, D. G., Golightly, M. G. and Koren, H. (1982) in Natural Killer Cells and Other Natural Effector Cells (Herberman, R. B., ed.), pp. 159-164, Academic Press, New York

20 Roder, J. C., Kiessling, R., Biberfeld, P, and Andersson, B. (1978)J. Immunol. 121, 2509-2517

21 Perlman, P. and Holm, G. (1969) Adv. lmmunoL I1, 117-193 22 Sanderson, C. J. and Taylor, G. A. (1975) Cell Tissue Kiuet. 8, 23-32 23 Thorn, R. M. and Henney, C. S. (1977)J. Immunol. 119, 1973-1977 24 Sullivan, K. A., Berke, G. and Amos, B. (1972) Transplantation 13,

627-628 25 Heitzmann, H. and Richards, F. M. (1974)Proc. NatlAcad. Sci. U.S.A

71, 3537-3541 26 Grimm, E. A., Price, Z. and Bonavida, B. (1979) Celllmmunol. 46, 77-99 27 Zagury, D., Bernard, J., Jeannesson, P., Thiernesse, N. and Cerottini,

J.-C. (1979)J. Immunal. 123, 1604-1609 28 Bradley, T. P. and Bonavida, B. (1982) in Natural Killer Cells and Other

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Natural Effector Cells (Herberrnan, R. B., ed.), pp. 145-152, Academic Press, New York

29 Bradley, T. P. and Bonavida, B.J. ImmunoL (in press) 30 Landazuri, M. O. and Herberman, R. (1972) Nature (London) New Biol.

238, 18-19 31 Schick, B. and Berke, G. (1979) Transplantation 27, 365-368 32 Martz, E. (1977) Contemp. Top. lmmunobiol. 7, 245 33 Fan, J., Ahmed, A. and Bonavida, B. (1980)J. ImmunoL 125, 2444-2453 34 Hiserodt, J. C. and Bonavida, B. (1981)J. Immunol. 126, 256-262 35 Effros, R. B., Hiserodt, J. C. and Bonavida, B. (1980)J. l m ~ l . 125,

1879-1884 36 Wexler, H., Fan. J., Hiserodt, J. C. and Bonavida, B. Cell Immunol. (in

press) 37 Klein, M., Roder, J., Haliotis, T., Koreu, S., Jett, J., Herberman,

R. B., Katz, P. and Fauci, A. S. (1980)J. Exp. Med. 151, 1049-1058 38 Roder, J. and Durve, A. (1979) Nature (London) 278, 451--453 39 Timonen, T., Ortaldo, J. R. and Herbermen, R. B. (1981)J. Exp. Med.

153, 569-582 40 Reynolds, C. W., Timonen, T. and Herberman, R. B. (1981)J. Immm~l.

127(1), 282-287

The Restless Tide: The Persistent Challenge of the Microbial World

by Richard M . Krause, National Foundation for Infectious Diseases, 1981. $1 6 .0 0 ( i + 152 pages) Library of Congress No. 82-116089

There is no doubt about the urgent im- portance of good 'popular science' writing. In a world of half-baked scientific ideas dominated by news values, real science would seem to throw from time to t ime a flickering light on what may in ' this pragmatical pig of a world ' be loosely considered truth. I cannot but believe that the ability to dis- tinguish truth from falsehood, which Socrates went on about so, is socially use- ful, and can be best learnt from the unders tanding of genuine scientific thought processes. I also believe that it would be desirable for all our masters, from newspaper proprietors and military. dictators to borough council officials and shop stewards, to achieve this ability. Those are the people to whom popular science writ ing should be addressed. Its point is not to demonstrate how clever scientists are, in their remote world of fundamental particles and algebraic formulae and wonder drugs, but to show that there is a continuous thread, and that the imagination is exercised in the same way, from the problems dealt with by Einstein or Crick to those such as how to fell a tree without damaging the rest of the wood, how to locate the source of the ' funny noise' which one ' s spouse has complained of in the car engine, or where in the oven to bake a meringue. It is positively bad for non-scientists to be told about science if they do not learn this too. Everyone can think, more or less: science

is just workaday thinking with a few heuristic rules.

Most popular writing about science is purely informative with all the scientific, i.e. critical, thinking simplified out of it; the analytical structure is given but not how it was derived, which is thought to be too 'difficult ' for the man-in-the- street to understand: if you despise your audience you get the audience you deserve. Every schoolteacher knows now that this is bad teaching technique; our schoolchildren are better instructed than the general public. There is also a re- quirement for a personal voice: as Eric Ashby says 1, the scientific writer should 'convey delight like a kitten playing with a ball of wool ... should tell you a lot about the essayist, his prejudices, his en- thusiasms, what he stands f o r . . . ' , qualities which have to be bleached out of technical scientific writing. This 'mandar in ' style of professional writing is a bad model for the popular science writer, professional though he must be.

O n all these criteria I feel that Krause ' s book does not really hit the jackpot. It is easy reading, it is informa-. tive and urbane, factually sound and convincing. Why therefore does one have a prevailing sensation of disap- pointment , of d~3~-vu? Nowhere alas is there an intrusive passion or prejudice, some abrasive opinion to make the reader sit up; anxieties once raised, about overpopulation for example or genetic engineering, are too often soothed by an emollient and woolly optimism - just the subjects upon which public anxieties, however unjustified, need to be taken seriously.

There is also a distressing lack of detail, or detail runs out just where one is getting interested. For example . . . ' In- tensive research is now focused on the manne r in which these genetic factors predispose to the allergic state'. 'Another area of investigation concerns . . . ' etc. Well, what is that intensive research? It is

not that Krause does not know: apart from being Director of the National Institute of Allergy at Bethesda, he has been personally associated with some of the most elegant research of recent years. It is because the general public must not be exposed to something still speculative and uncertain, poor things. All the best popularizers of science of the modern period, such as J . B. S. Haldane and Lewis Thomas , have not hesitated to deal with the shadowy frontiers of advancing knowledge, and to adduce their own ideas and speculations; Krause is too cautious, or too humble, to present any idea which has not the well-rubbed patina of the market-place. This is a pity, since apart from anything else it is positively dangerous for science to be made to sound too certain and too peace- ful rather than a structure of militant hypotheses all of which one expects to discard within ten or twenty years. Otherwise the public will accept (as it does) a pronunciamento by Dr This or Professor That , as if it were a truth written with a plume plucked from the wing of the angel Gabriel. Krause, to be fair, does describe a few blind alleys of the past and perplexities as to the future; this is by no means just a flashy journalis- tic description of scientific break- throughs. So we have a good description of microbial resistance mechanisms, of the contribution of the magnificent and still underest imated Oswald T. Avery to molecular biology, on heart disease, bilharzia, malaria. This book is strong on information, but not quite strong enough on ideas.

Reference 1 Ashby, E. (1980) Nature (London) 284, 193

P. G. H. GELL

P. G. H. Gell is professor in the Department of Pathology, University of Cambridge, Cambridge CB2 1QP, UK.