949

the genetic message could be "tattooed" into fixed histiocyteswithin a mastectomy wound, to announce themselves later as "localrecurrences".The integration of RNA-directed, retroviral genomic DNA into

the host cell genome is an obligatory step in the retroviral life cycle.3It is not beyond the realms of possibility that subsequenttranscription of the retroviral DNA copy, perhaps many cell

generations later, leads to the assembly of retroviral particles withoncogenic potential. Distant metastases which appear many yearsafter the primary tumour could be caused by the integration ofretroviral genes adjacent to those cellular genes which normallyencode the breast phenotype. Retroviral promotor elements maylead to the activation of these genes, resulting in a breast-derivedsecondary within another organ.

Alternatively the transfection process may result in the stableintegration of large sections of breast cancer derived DNA which, ifinserted in the correct orientation and if subsequently "fired" bysuitable promotor sequences, could ultimately lead to a secondarytumour which expresses breast phenotypic markers. These largesections of genomic material could be conserved by the monocyte-macrophage system, as outlined above, in such a way that its

transmission, transfection, and expression might lead to metastasesthat are distant in both time and location.We do not challenge the hypothesis that most distant metastases

result simply from a mechanical transfer of cells from the primaryfocus to distant sites but we suggest that there may be twomechanisms for the initiation of distant metastases-the earlier ones

resulting from the classical mode of dissemination and the remotevariety resulting from a transfection-like process. If so, we mightbetter understand the disappointments of adjuvant systemicchemotherapy delivered within 12 months of primary surgery.’

Department of Surgery,Kings College School of Medicineand Dentistry,

Rayne Institute,123 Coldharbour Lane,London SE5 9NU

MICHAEL BAUMANTHONY COLLETTASTEPHEN R. EBBS

1 Baum M. The curability of breast cancer. In: Stoll BA, ed. Breast cancer management:Early and late. London Heinemann, 1977: 3-13.

2. Holt S, Croton R, Lemster SJ, et al. In vitro thymidine labelling index in primaryoperable breast cancer. Eur J Surg Oncol 1986: 12: 53-57

3. Bishop JM. Cellular oncogenes and retroviruses. Annu Rev Biochem 1983; 52: 301-54.4 Wilson AJ, Houghton J, Baum M. Adjuvant therapy for breast cancer. In: Slevin ML,

Staquet MJ, eds. Randomized trials in cancer: a critical review by sites. New York:Raven Press, 1986 359-84.

CARBON DIOXIDE, BRAIN DAMAGE, AND CARDIACSURGERY

SIR,-Dr Gilston and Mr Crockett (Feb 13, p 353) assert thatboth the title and conclusion of our paper (Dec 26, p 1493) areunjustified. It is vital to clarify our findings and dispel some of themisapprehensions that Gilston and Crockett’s letter might raise.We did not imply a causal relation between pre-bypass

hypocapnia and hypocapnia or hypercapnia after the onset ofbypass, but an obvious temporal relation does exist.

In most centres doing cardiopulmonary bypass (CPB) surgery inthe UK it is unusual to do more than two blood-gas analyses beforethe onset of bypass (paediatric cases excepted). Nearly all thesesamples are taken soon after the patient has been placed on aventilator at the beginning of surgery. As dissection may takeanything up to one hour, in the case of internal mammary grafts, weare dependent on a subjective assessment of the patient’s ventilatoryrequirements for the maintenance of normocapnia. Our studyshowed that if a widely accepted formula for the calculation of theserequirements is applied (tidal volume 7-5 ml/kg, rate 12/min) thenover 75% of the subjects were hypocapnic on entering the bypassphase. In addition, during the CPB, continuous monitoring showedlarge fluctuations in individual PaC02 measurements (often relatedto periods of reduced flow) that would obviously be missed ifblood-gas was analysed only every 30 min.We did not set out to suggest the "ideal" method for monitoring

during bypass but rather to observe the practices of a large centre,and to see if improvements were needed. Gilston and Crockett’s

comment, therefore, on the disadvantage of radial arterial pressuremonitoring is somewhat superfluous as this is the only arterial sitethat is routinely monitored in the UK. More importantly what wethought vital to highlight was high cerebral venous pressures ofsuch an order, in many of the subjects, to have a significant bearingon the overall cerebral perfusion pressure, whatever the source ofthe arterial monitoring.With regard to intergroup discrepancies in the duration of CPB,

only 7 patients out of a total of 65 had bypass periods of longer thantwo hours. Of these only 4 were in the uncontrolled PaC02 group;the bypass times for the remaining members of both groups wereotherwise similar with mean bypass times of 83 and 87 min,respectively. While we did see significant deficit with the longerbypass times this did not represent a major difference between thetwo groups.

Gilston and Crockett say that hyperventilation is routinely usedin both general anaesthesia and intensive care without evidence ofpostoperative brain impairment. The deficit we describe is muchmore diffuse than classical "stroke". It is represented by subtlechanges in a wide variety of cognitive skills which, althoughdistressing to patients and their relatives and having majorimplications on functional capabilities, may not be immediatelyobvious in a busy clinical setting.The preliminary findings of our further study, looking at the

incidence of neurological deficit in patients undergoing arterialsurgery without CPB (unpublished), together with the studies ofhigh altitude climbers cited by Professor West (Feb 13, p 353), addweight to the beliefs of those such as Dr Feldman (Feb 13, p 353)that hyperventilation is not as safe as Gilston and Crockett mighthave us believe.

In view of the well-established relation between PaC02 andcerebral arteriolar tone it is feasible that hypocapnic constriction ofreactive vessels and a further reduction in cerebral blood flow withthe onset of bypass will be important contributors to the productionof postoperative neurological deficit.

Department of Cardiothoracic Surgery,St Georges Hospital,London SW17 0QT

MICHAEL NEVIN

JOHN R. PEPPER

BLUE DYES IN RECONSTRUCTIVE SURGERY

SIR,-Surgeons involved in cutaneous reconstruction have, formany years, planned their incisions by marking with a pen and inkbefore taking the irrevocable step of cutting the skin. Most havefound Bonney’s blue1 to be the most satisfactory dye. It does notpermanently tattoo the skin; and it withstands wetting with blood orsaline yet may be easily erased with spirit. This practical experiencehas been supported by experimental studies.2

In 1987 the UK Government’s Food Advisory Committee3 3recommended that gentian-violet (which is contained in Bonney’sblue) should not be used to mark citrus fruits. Long-term systemicabsorption studies had revealed an increase in liver cell tumours andreticulum cell sarcomas when mice were fed a 99 % gentian-violet/1 % methyl-violet preparation.’ As a result the Department ofHealth has advised manufacturers that such preparations shouldnot be used on mucous membranes or open wounds. ThePharmaceutical Society in its turn advised pharmacists that if theyreceive a prescription for gentian-violet paint, they should contactthe prescriber to check that he is aware of these concerns before theydispense it. In practice many hospital pharmacies have withdrawngentian-violet and have told doctors that it is no longer available.This leaves surgeons with the choice of using an inferior dye such asmethylene-blue (which invariably becomes invisible before anyincision is made) or a fibre-tipped marking pen, which is too coarsefor procedures such as cleft lip repair, where an error of 05 mm maybe disastrous. It may be difficult to appreciate the importance of thisproblem without watching the surgery involved.We do not wish to cast doubt on the validity of the mouse studies

but is this really a sensible application of experimental evidence to aclinical activity? There is a world of difference between the

application of a drop or two of a subtance to the skin, and along-term diet of high concentrations of the same substance. Many

950

thousands of patients have had their skin marked with Bonney’sblue and to our knowledge there has been no report of any inducedmalignancy, hepatic or otherwise. Cannot pharmacists interpretrecommendations more rationally and distinguish between its use inlarge quantities on open wounds or mucous membranes and as amarking ink? There is no satisfactory alternative.

Craniofacial Centre,Hospital for Sick Children,London WC1N 3JH

BARRY M. JONESDAVID T. GAULT

1. Johnson HA. "Bonney’s blue marking ink. Plast Reconstr Surg 1975; 56: 155-56.2. Granick MS, Heckler FR, West-Jones E. Surgical skin marking techniques. Plast

Reconstr Surg 1987; 79: 573-80.3. Food Advisory Committee. Final report on the review of the colouring matter in food

regulations 1973. London: HM Stationery Office, 1987.4. NCTR. Chronic toxicity and carcinogenicity studies of gentian violet in mice. (NCTR

Tech Rep): Experiment 304. Jefferson, Arizona. National Center for ToxicologicalResearch, 1984.

RECOMBINANT HUMANGRANULOCYTE-MACROPHAGE

COLONY-STIMULATING FACTOR PRODUCESFEVER

SIR,--Colony-stimulating factors promote the proliferation anddifferentiation of haemopoietic precursors and, in some cases, affectthe function of mature peripheral effector cells.12 Human

granulocyte-macrophage colony-stimulating factor HuGM-CSF isa 14-35 kD glycoprotein produced by endothelial cells, fibroblasts,and T cells and stimulates myeloid proliferation anddifferentiation.3 This growth factor has been purified, cloned,expressed in vitro, and administered to manyWe have shown that continuous intravenous administration of

recombinant HuGM-CSF after high-dose alkylating agent

Temperature charts for two patients given rHuGM-CSF.

(A) without ibuprofen; (B) with ibuprofen.

chemotherapy and autologous bone marrow support results inmyeloid bone-marrow maturation, accelerated granulocyterecovery, and reduced infections and treatment-related toxicity.4 4The final evaluation of the role of this agent as an adjunct tochemotherapy requires a randomised clinical trial. Endpointssuggested for such trials include number and type of infections anddays of fever or fever and neutropenia.As part of a phase II trial, we administered daily 4 h infusions of

rHuGM-CSF for 21 days after high-dose chemotherapy andautologous bone-marrow infusion to fourteen patients with breastcancer and melanoma.

Figure A shows the oral temperature chart for a patient receivingno transfusions or medications other than rHuGM-CSF.Beginning with the start of the rHuGM-CSF infusion, the

temperature rose to a peak at or shortly after the end of the infusion.Discontinuation of the rHuGM-CSF infusion was associated withthe disappearance of fever. Figure B shows oral temperatures of asecond patient receiving rHuGM-CSF in a similar manner. In thispatient, premedication with a single dose of ibuprofen (400 mgorally) appeared capable of blocking fever development associatedwith the rHuGM-CSF; discontinuation of ibuprofen was

associated with febrile reactions to rHuGM-CSF infusion. Finally,with discontinuation of rHuGM-CSF after day 21, fever resolved.These data provide clear evidence that the administration of

rHuGM-CSF can be associated with the development of fever andsuggest that the fever is at least partly mediated throughprostaglandin pathways since it is blocked by ibuprofen. Themechanism of fever production may be complex and mediatedthrough rHuGM-CSF induced production of other endogenouscytokines, such as interleukin-1 or tumour necrosis factor, or byother mechanisms. Studies to evaluate these possibilities are inprogress.The clear association of fever with drug administration suggests

caution in the use of fever as an endpoint in clinical trials of

rHuGM-CSF. Further, although ibuprofen appears capable ofameliorating this fever, we are concerned that the routine use ofnon-steroidal anti-inflammatory medication may be associated withrenal dysfunction, or that the non-specific suppression of fever maydelay detection of significant infections.

Bone Marrow Transplant Program,Duke University Medical Center,Durham, North Carolina 27710, USA

WILLIAM P. PETERS

JEFFREY SHOGANELIZABETH J. SHPALLROY B. JONESCHUL SOO KIM

1. Metcalf D. The hemopoietic colony stimulating factors. Amsterdam Elsevier, 1984.55-96.

2. Sieff CA. Hematopoietic growth factors. J Clin Invest 1987; 70: 1549-57.3. Groopman JE, Mitsuyasu RT, DeLeo MJ, et al. Effect of recombinant human

granulocyte-macrophage colony-stimulating factor on myelopoiesis in the acquiredimmunodeficiency syndrome. N Engl J Med 1987; 327: 593-98.

4. Brandt SJ, Peters WP, Atwater SK, et al. Effect of recombinant human granulocytemacrophage colony-stimulating factor on hematopoietic reconstitution after

high-dose chemotherapy and autologous bone marrow transplantation. N Engl JMed (in press).

5. Vanhan-Raj S, Keating M, LeMaistre A, et al. Effects of recombinant human

granulocyte macrophage colony-stimulating factor in patients with myelodyspasticsyndromes. N Engl J Med 1987; 317: 1545-52.

YAWNING

SIR,-Dr Wimalaratna and Dr Capildeo (Feb 6 p 300) assert thatthe purpose of yawning remains a mystery and Dr Forrester (March12 p 596) suggests that its purpose is to maintain lung complianceand inflate collapsed alveoli. I suggest a third possibility. AsWimalaratna and Capildeo mention, yawning is "infectious".Animals would have a greater tendency to sleep together if the firstto become weary was to signal and if the others were to respond.The infectious nature may therefore have a group cohesive

function, which has survival implications for animals that hunt inpacks or protect themselves in herds.

Friern Hospital,London N11 3BP MALCOLM P. I. WELLER