Gut, 1979, 20, 833-839

Suppressive effect of alcoholic liver disease sera onlymphocyte transformation1G. P. YOUNG,2 F. J. DUDLEY,3 AND M. B. VAN DER WEYDEN

From Monash University Department of Medicine, Alfred Hospital, Victoria, Australia

SUMMARY The effect of alcoholic patient sera on in vitro lymphocyte transformation was studiedusing mitogen-induced uptake of 3H-thymidine to measure blastogenesis. With pokeweed mitogenas the stimulus, transformation of normal lymphocytes in sera of alcoholics with either normal orfatty livers was not significantly different from that obtained in pooled human serum (PHS).However, in sera of patients with either alcoholic hepatitis or inactive cirrhosis mean transformationwas significantly reduced (P <0001, <0*02 respectively). With phytohaemagglutinin-P or con-canavalin A as mitogens, suppression of transformation was not as marked but followed the samepattern. A significant negative correlation was observed between the magnitude of transformationand serum bilirubin and aspartate aminotransferase levels. An intra-patient comparison of the effectson transformation of normal lymphocytes by simultaneously collected peripheral and portal venoussera, and of peripheral sera obtained before and after portasystemic shunt surgery, indicated thatthe factor(s) responsible did not originate in the splanchnic circulation nor did it accumulate in theserum because of failed hepatic clearance. By performing transformation experiments in the presenceof inhibitory patient sera diluted with PHS it was possible to show that these sera caused trueinhibition of transformation rather than suppression due to failure to sustain cell culture becauseof nutritional deficiencies. Inhibitory sera did not contain high levels of the enzyme thymidinephosphorylase and did not significantly inhibit binding of 125I-labelled mitogens to the lymphocytesurface. These findings indicate that the inhibitory effect of sera from alcoholics is of potentialin vivo importance, that the effect increases with the degree of heptocyte damage, and that it isunrelated to the nonhepatic metabolic affects of chronic alcoholism.

Although it has been demonstrated that the sera ofpatients with a variety of hepatic disorders includingviral hepatitis, primary biliary cirrhosis, chronicactive hepatitis, and alcoholic liver disease cansuppress in vitro lymphocyte transformation (Mellaand Taswell, 1970; Hsu and Leevy, 1971; Fox et al.,1973; Newberry et al., 1973; Macsween and Thomas,1973; Wands et al., 1975; Nakao et al., 1975;Brattig and Berg, 1976), the nature of thisphenomenon is poorly understood in the majority'This study was supported by the Alfred Hospital HenryLaurie Scholarship Fund.2G. P. Young was supported in part by a Royal AustralasianCollege of Physicians research grant. Present address:Division of Gastroenterology, Washington University Schoolof Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110,USA.3Address for reprint requests: Dr F. J. Dudley, Gastro-enterology Diagnostic Service, Alfred Hospital, CommercialRoad, Prahran, Victoria 3181, Australia.

Received for publication 6 June 1979.

of instances. In chronic alcoholic patients it is notclear whether this effect is a consequence of liverdamage or of the accompanying metabolic effects ofalcoholism, including nutritional deficiency. Thisinhibitory effect of serum might also be an artefactin that it may be peculiar to the conditions ofculture and hence of no possible relevance to in vivolymphocyte function. Furthermore, existing reportsof this phenomenon in alcoholic liver disease areconflicting and do not relate it to the spectrum ofalcoholic liver damage (Hsu and Leevy, 1971;Newberry et al., 1973; Thestrup-Pedersen et al.,1976).

In an attempt to define the incidence andcharacteristics of this immunosuppressive phenome-non we have analysed the effect of sera from chronicalcoholics, with or without liver disease, on mitogen-induced transformation of normal lymphocytes. Inaddition, we attempted to identify the source of thefactor(s) responsible for this suppression and ex-

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G. P. Young, F. J. Dudley, and M. B. Van der Weyden

amined the possibility that this effect might be anin vitro artefact of no in vivo significance.

Methods

PATIENTSHospitalised patients suffering from chronicalcoholism, defined as a daily consumption of morethan 100 g of alcohol for 10 years, were consideredfor admission to the study. Patients suffering fromuraemia, pregnancy, uncorrected nutritionaldeficiency, suspected or confirmed autoimmuneor viral liver disease, severe infection, neoplasia,or those receiving immunosuppressive therapy wereexcluded. Of those included, 39 were hospitalisedbecause of suspected liver disease and the remainderfor non-hepatic complications of chronic alcoholism.On the basis of clinical and biochemical findings andliver histology these patients have been arbitrarilydivided into four groupsI Alcoholics: normal liver function with normal

histology or fatty change only on liver biopsy (10patients).

2 Alcoholic hepatitis: defined as patients withhistological or biochemical evidence of alcoholichepatitis (Sherlock, 1975), with or withoutcirrhosis (14 patients)

3 Alcoholic cirrhosis: defined as patients withfeatures of alcoholic cirrhosis (Sherlock, 1975)without evidence of hepatitis (15 patients).

4 Alcoholic cirrhosis and a surgical portasystemicshunt (12 patients).Serum was obtained from each patient by vene-

puncture and then stored under sterile conditions at-24°C. Normal human pooled serum (PHS) wasobtained from 100 normal blood donors and storedunder identical conditions. Peripheral and splanchnicvenous sera were simultaneously obtained from fourpatients with alcoholic cirrhosis undergoing porta-systemic shunt surgery and from four non-alcoholicpatients with normal liver function undergoingcholecystectomy. Approval to carry out this studywas obtained from the Alfred Hospital ResearchAdvisory Committee.

LYMPHOCYTESCitrated peripheral venous blood samples, obtainedfrom 18 healthy medical students and laboratorypersonnel, were centrifuged at 170 g for 20 minutesand the platelet rich plasma discarded. The buffycoat was suspended in Hanks' balanced salt solution(HBSS) and the mononuclear cells isolated by Ficoll-Hypaque gradient centrifugation (Boyum, 1968).After centrifugation at 800 g for 20 minutes thelymphocyte rich interface was removed and washed

twice with HBSS. The cell pellet was suspended inEagle's minimum essential medium (MEM), incubat-ed at 37°C for one hour in a 5% C02/air mixture andwashed once with MEM. The final cell preparationhad a platelet to mononuclear ratio of approximately4:1 with a cell viability, as determined by trypan blueexclusion, of greater than 980%. On stained smearsof the lymphocyte preparations more than 900% ofthe cells were lymphocytes. For mitogen bindingstudies the mononuclear preparations obtained inthis way were centrifuged through a discontinuoussucrose gradient (Perper et al., 1968) reducing theplatelet to mononuclear ratio to 1:4.

E-ROSETTESE-rosettes were measured in the presence of 20%PHS or patient serum according to Chisari andEdgington (1975). The lymphocyte :sheep erythrocyteratio was 1 :40.

LYMPHOCYTE STIMULATIONLymphocyte stimulation was performed in microtitreplates containing 1 25 x 105 normal donor cells perwell in 0-26 ml of MEM containing penicillin andstreptomycin, 100 units/ml, and supplemented with20% patient or PHS. All tests were performed intriplicate and the mitogens used were purifiedphytohaemagglutinin (PHA-P; CSL, Melbourne),pokeweed mitogen (PWM; Grand Island BiologicalCo., New York), and concanavalin A (Con A;Calbiochem, Sydney). The concentrations of PHA-Pused were 11 5, 46, and 185 ,g/ml; PWM, 1, 4, and10 tg/ml; and Con A 10 .tg/ml. The cultures wereincubated at 370 for 72 hours in a humidified 5%C02/air atmosphere. Four hours before the end ofthe culture period, 0 5 ,uCi of (methyl-3H)-thymidine(The Radiochemical Centre, Amersham, 5 Ci/mmolwas added to each well in 0-01 ml of the culturemedium. Trichloroacetic acid (TCA) precipitablematerial was collected on glass fibre filters byharvesting the cells with the aid of a multiple auto-mated sample harvester (Titertek, Skatron, Norway)and radioactivity was determined in a Packardliquid scintillation counter. Results were expressedas mean cpm of triplicate cultures. The resultsobtained in 20% PHS for each batch of lymphocytesacted as the control. Patient groups were comparedwith controls using Student's t test (paired).

LYMPHOCYTE-MITOGEN BINDINGCon A and PWM were iodinated with 1251 (Na'251,Australian Radiation Laboratory) using the lacto-peroxidase method (Phillips and Morrison, 1970).Both compounds retained their mitogenic propertiesafter this procedure. The labelled mitogens, atconcentrations used in the transformation experi-

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Lymphocyte transformation in sera from alcoholics

ments, were added to lymphocytes suspended inMEM and 20% human serum. After incubation atroom temperature for 15 minutes the cells werecentrifuged at 1000 g for two minutes and washedtwice with phosphate buffered saline. All experimentswere performed in triplicate, and in the case of 1251.Con A, with and without 0-2 M x methyl-D-glucopy-ranoside (a MG). 1251 binding was determined in aNuclear Chicago gamma spectrometer. Results wereexpressed as cpm/4 x 105 cells, the binding ofmitogen to the plastic incubation tubes being

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subtracted in all cases. In the case of 125I-Con A, a

MG inhibited binding to cells by up to 90% and theeffect of serum on this specific binding only wasconsidered.

THYMIDINE PHOSPHORYLASE ASSAYThese were carried out under the conditions appliedin lymphocyte culture using a modified microassaytechnique (Pauly et al., 1977) in which the chroma-tography papers were developed by ascendingchromatography. Conversion of tritiated thymidine

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Fig. 1 Transformation of normal lymphocytes measured by 3H-thymidine uptake by trichloroacetic acid precipitatesin individual test sera using optimal dosage of (A) pokeweed mitogen and (B) phytohaemagglutinin-P. Solid horizontalbars represent the mean for each group and the dotted lines the standard error of the mean. P values refer tocomparison between alcoholic sera sub-groups and pooled human serum ('controls') (see text). 'Alcoholics' refersto chronic alcoholics with normal or fatty livers.

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G. P. Young, F. J. Dudley, and M. B. Van der Weyden

to tritiated thymine was determined by measuringradioactivity in the appropriate areas by liquidscintillation counting. Enzyme activity was expressedas the percentage tritiated thymidine degraded totritiated thymine.

Results

The effects of sera from patients suffering fromchronic alcoholism on PHA - or PWM - inducedtransformation of normal peripheral blood lympho-cytes are shown in Fig. 1. With optimal concentrationof PWM (Fig. la), transformation of normallymphocytes in sera from patients with alcoholichepatitis (4526±1051 cpm/1 25x 105 lymphocytes,mean ±SE, P <O 001), alcoholic cirrhosis (10457 ±1772 cpm, P <0 05), and post-shunt alcoholiccirrhosis (10595±2004 cpm, P<0-05) was signifi-cantly reduced compared with normal controls(16469 ± 1508 cpm). In contrast, sera from alcoholicswith normal liver function did not suppress trans-formation (17337 ± 1134 cpm). Using PHA-Psuppression was less marked than with PWM (Fig.lb). In non-shunted cirrhotics suppression withPHA-P was significant when compared with alcoho-lics with normal liver function (p <0-05) but notwhen compared with control PHS (0 1O> P> 005).In data not shown here, the same pattern of responsewas obtained using optimal concentration of Con A.Unstimulated cultures in PHS gave 134±75 cpm( ±SE) and did not differ significantly from results inpatient sera.The observed inhibitory effect of sera on transfor-

mation was independent of the duration of culture,for similar findings were obtained in experiments inwhich cells were harvested three, four, and five daysafter mitogen stimulation. Supraoptimal concentra-tions of PHA-P or PWM failed to overcomeobserved suppression in the large majority ofpatients (Fig. 2). Transformation in sera from thegroup of cirrhotic patients with surgical porta-systemic shunts did show significant improvementwith increasing doses of PHA-P, although this didnot reach a significantly higher level than transfor-mation with supraoptimal doses in sera fromcirrhotics without surgical shunts.A significant degree of negative correlation was

observed between the magnitude of transformationusing either mitogen and the serum bilirubin level(r=-0-5138, P<0 001 for PHA-P; r=-0 4909,p < 0.01 for PWM), between serum aspartate amino-transferase levels and PWM-induced transformation(r= 0-3301, P <0 01), and between y-globulinlevels and PHA-P-induced transformation (r==-0-3650, P <0-05). No significant correlation(positive or negative) could be demonstrated

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Fig. 2 Mean phytohaemagglutinin (PHA-P) stimulatedtransformation of normal lymphocytes in sera from eachsubgroup of chronic alcoholics and in pooled humanserum (PHS). Vertical bars represent standard error ofthe mean. Alcoholic hepatitis 0; alcoholic cirrhosis 0;post-shunt cirrhotics V; PHS A; alcoholics without liverdisease E.

between the magnitude of transformation andserum levels of albumin, alkaline phosphatase,suppression of E-rosette formation, fibrin degrada-tion products, and very low density lipoproteins.No patient was x-foetoprotein positive.

EFFECT OF PORTACAVAL SHUNTING ONSERUM INHIBITIONPeripheral blood sera obtained from nine patientswith cirrhosis and portal hypertension just before andone month after portasystemic shunt surgery werestudied. With optimal concentration of PHA-P,transformation of normal lymphocytes (mean+SE)using preshunt sera was 19 102 ±3039 cpm andwith postshunt sera was 21 435 ±3848 cpm (p> 0-2).Results obtained with other concentrations ofPHA-P, and with PWM as the mitogen, confirmedthat the suppressing effect of these sera was notaccentuated by an increase in degree of porta-systemic shunting. All patients appeared to havepatent shunts at the time of study on the basis of

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Lymphocyte transformation in sera from alcoholics

clinical findings and scintisplenoportography insome patients.

In either controls or alcoholic cirrhosis patients,comparison of the effects of corresponding portalversus peripheral sera on transformation revealedno significant differences.

MECHANISM AND NATURE OF INHIBITIONBY PATIENT SERA

Thymidine phosphorylase assaysThymidine phosphorylase activity was measured inPHS and 11 suppressing sera. Mean enzyme activity(±SE) in 20% PHS was 14-0±1-0%. The meanlevel in 200% patient sera was 9-0 ±I.1 0% (range1 4-278 0%) with only one patient having a meanvalue in excess of that for PHS.

Lymphocyte-mitogen bindingEleven patient sera most suppressive for Con A-induced transformation (mean suppression of 40-4 %)failed to cause a significant reduction of specific ConA binding-mean binding in control PHS was5713±429 cpm/4x 105 cells (±SE) against 5369±428 in patient sera. Similarly, eight sera suppressivefor PWM (mean suppression of 57.2 %) gave a meanbinding of 2095±180 cpm/4x 105 cells (±SE)against 2597 ±407 in PHS. These differences are notsignificant.

Effect of dilution and dialysisTo ascertain if the inhibitory effect of patient serawas principally a consequence of a nutrient defi-ciency, transformation experiments were simul-taneously performed in two sets of culture conditionswhich were then compared: (1) PHS only in thecultures, in proportions of 5 %, 10 %, 15 %, and200%; (2) PHS mixed with individual patient sera inratios of 3:1, 2:2, 1:3, and 0:4 respectively, the totalserum concentration being maintained at 20 %. Theresults are shown in Fig. 3. Using sera from fivepatients, with PWM as the mitogenic stimulus, theaddition of 10% or 5% patient serum to 10% or150% PHS respectively, reduced transformation.Seven patients were similarly tested with PHA-P andfive with Con-A and similar patterns were observed.

Dialysis of sera against culture media did notreduce their inhibitory effect and heating to 56°C forone hour did not significantly alter the degree ofinhibition of transformation.

Discussion

Previous reports of the effect of sera from patientswith alcoholic liver disease on lymphocyte transfor-mation are conflicting. Whereas Hsu and Leevy(1971) and Newberry et al. (1973) found that sera

from patients with alcoholic cirrhosis suppressed invitro lymphocyte transformation, a more recentstudy using heat-treated sera (Thestrup-Pedersen etal., 1976) failed to find such an effect. In the presentreport it has been demonstrated that the sera ofpatients with chronic alcoholism have wide-rangingeffects on lymphocyte transformation, possiblyexplaining these conflicting reports. In addition, wefound significant inhibition to occur only in serafrom those with cirrhosis or alcoholic hepatitis,suggesting that alcoholic liver damage is responsiblerather than nonhepatic metabolic effects of alco-holism. The slight possibility that suppressionresulted from failure to correct subtle nutritionaldeficiencies despite clinical and biochemical nor-mality seems unlikely, as the results of serumdilution experiments (Fig. 3) can be explained onlyby the presence of an active inhibitor(s) of tritiated-thymidine uptake.The mechanism by which these sera cause sup-

pression remains obscure but a number of alter-natives peculiar to in vitro conditions, and thus ofno in vivo consequence, have been excluded. Theenzyme thymidine phosphorylase has been shown tobe present in plasma and has prompted the sugges-tion that alteration in its activity may lead toerroneous interpretation of these effects of differingsera on lymphocyte transformation (Pauly et al.,1977). However, assay of this enzyme in control and

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PER CENT PHS IN CULTURE

Fig. 3 The effect on pokeweed mitogen-inducedlymphocyte transformation of varying percentages ofpooled human serum (PHS) only (0-0) or mixing withpatient serum (0-0) in varying proportions to keeptotal serum concentration at 20 %. The patient sera curve( 0-0) represents the mean for five different sera.Vertical bars represent SE of the mean.

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838 G. P. Young, F. J. Dudley, and M. B. Van der WeydeiTinhibitory sera demonstrated that changes in itsactivity could not account for the observed sup-pression. It is also possible that sera may interferewith mitogen-lymphocyte interaction by causingsteric hindrance with surface receptors for mitogen(Boldt et al., 1972; Nicolson, 1974) or by serumprotein binding of large amounts of the mitogenicstimulant (Humphrey et al., 1974). These effectsappear unlikely in this study, however, as 1251-PWMand 1251-Con A binding experiments showed thatquantitative mitogen binding to lymphocytes wasnot significantly reduced by inhibitory sera; manyof the suppressing sera suppressed transformation toall three mitogens, each having different receptorspecificity (Nicolson, 1974); and with only occa-sional exceptions could inhibition be overcome byincreasing the dose of mitogen.As inhibition is most marked in sera from patients

with alcoholic hepatitis, and as it correlates withbilirubin and AST levels, hepatocyte damage appearsimportant in the development of inhibitory factorsin the serum (SIF). It is not clear whether SIF isreleased from damaged hepatocytes or not. Theeffects of peripheral and portal venous sera ontransformation were similar for both patients andnon-alcoholic controls, and, as SIF did not increaseafter portasystemic shunt surgery, it is unlikely thatSIF originates in the splanchic circulation and gainsaccess to the peripheral circulation either because offailed clearance by the damaged hepatocyte orbecause of portasystemic shunting.

In hepatic cirrhosis failure of hepatic clearance ofgut antigens results in an increased antigenicchallenge, which appears to be a major factorleading to the hypergammaglobulinaemia seen inmany patients (Zinneman, 1975). In vitro animalstudies have shown that sensitised lymphocytesexposed simultaneously to multiple antigens, pro-duce soluble factors inhibitory to antibody produc-tion (Kempf and Rubin, 1977) and lymphocyteproliferation (Thomas et al., 1975) SIF may thus bea non-specific immunoregulatory factor released bylymphoid cells of patients with alcoholic liverdisease as a consequence of hyperstimulation of theimmune system.

These observations, that suppression of lympho-cyte transformation does not appear to result from(1) nutrient deficiencies in the sera, (2) increasedserum thymidine phosphorylase levels, or (3) inter-ference with lymphocyte-mitogen interaction, are ofsome importance. They suggest the possibility thatSIF acts at a point common to both lectin- andantigen-induced blastogenesis and thus could modifyin vivo function of lymphocytes with immunologicalconsequences. Both the susceptibility of alcoholics,particularly with liver disease, to various infections

(Straus and Berenyi, 1971, 1973; Berenyi et al., 1974;Leevy et al., 1976; Isselbacher, 1977) and the impair-ment of delayed sensitivity reactions observed inalcoholics with liver disease (but not in those withoutliver disease) (Berenyi et al., 1974) may be in part aconsequence of inhibition of in vivo lymphocytefunction by SIF. This effect of SIF on nucleotideincorporation may inhibit lymphocyte turnover andexplain the lymphopenia encountered in patientswith alcoholic liver disease (Young et al., 1979).The presence of SIF in the sera of subjects with

alcoholic liver disease represents a further metabolicabnormality in these patients, which may haveimportant immunological clinical consequences.Further characterisation of the nature of SIF, themechanism by which it affects lymphocyte function,and its in vivo consequences seems warranted.

We wish to thank Mr Ian Rose for his excellenttechnical assistance and Dr A. Goh and Dr J.Rolland for their helpful advice.

References

Berenyi, M. R., Straus, B., and Cruz, D. (1974). In vitroand in vivo studies of cellular immunity in alcoholiccirrhosis. American Journal of Digestive Diseases, 19,199-205.

Boldt, D., Skinner, A. M., and Kornfeld, S. (1972).Studies of two subpopulations of human lymphocytesdiffering in rasponsiveness to concanavalin A. Journalof Clinical Investigation, 51, 3225-3234.

Boyum, A. (1968). Isolation of mononuclear cells andgranulocytes from human blood. Scandinavian Journalof Clinical and Laboratory Investigation, 21, suppl. 97,77-89.

Brattig, N., and Berg, P. A. (1976). Serum inhibitoryfactors (SIF) in patients with acute and chronic hepa-titis and their clinical significance. Clinical and Experi-mental Immunology, 25, 40-49.

Chisari, F. V., and Edgington, T. S. (1975). Lymphocyte Erosette inhibitory factor: a regulatory serum lipo-protein. Journal of Experimental Medicine, 142,1092-1107.

Fox, R. A., Dudley, F. J., Samuels, M., Milligan, J., andSherlock, S. (1973). Lymphocyte transformation inresponse to phytohaemagglutinin in primary biliarycirrhosis: the search for a plasma inhibitory factor.Gut, 14, 89-93.

Hsu, C. C. S., and Leevy, C. M. (1971). Inhibition ofPHA-stimulated lymphocyte transformation by plasmafrom patients with advanced alcoholic cirrhosis.Clinical and Experimental Immunology, 8, 749-760.

Humphrey, G. B., Lankford, J., Oleinick, S., andNitschke, R. (1974). Leukemic serum inhibition ofphytohemagglutinin-induced transformation. Journalof Immunology, 113, 63-69.

Isselbacher, K. J. (1977). Metabolic and hepatic effects ofalcohol. New England Journal of Medicine, 296,612-616.

group.bmj.com on July 10, 2011 - Published by gut.bmj.comDownloaded from

Lymphocyte transformation in sera from alcoholics 839

Kempf, K. E., and Rubin, A. S. (1977). Transient sup-pression of the humoral immune response mediated bya factor derived from specifically activated, doublyprimed lymphoid cells. Journal of Immunology, 119,517-523.

Leevy, C. M., Chen, T., Luisada-Opper, A., Kanaga-sundarum, N., and Zetterman, R. (1976). Liver diseaseof the alcoholic: role of immunologic abnormalities inpathogenesis, recognition and treatment. Progress inLiver Diseases, 5, 516-530.

Macsween, R. N. M., and Thomas, M. A. (1973). Lymph-ocyte transformation by phytohaemagglutinin (PHA)and purified protein derivative (PPD) in primary biliarycirrhosis. Clinical and Experimental Immunology, 15,523-533.

Mella, B. A., and Taswell, H. F. (1970). Suppression ofleukocytic mitosis by sera of hepatitis-implicateddonors. American Journal of Clinical Pathology, 53,141-144.

Nakao, M., Mizoguchi, Y., Monna, T. et al. (1975).Studies on the sub-population and function of peri-pheral lymphocytes, and inhibitor to PHA stimulationexisting in the serum of patients with liver disease.Gastroenterologica Japonica, 10, 307-315.

Newberry, W. M., Shorey, J. W., Sanford, J. P., andCombes, B. (1973). Depression of lymphocyte reactivityto phytohemagglutinin by serum from patients withliver disease. Cellular Immunology, 6, 87-97.

Nicolson, G. L. (1974). The interactions of lectins withanimal cell surfaces. International Review of Cytology,39. 89-190.

Pauly, J. L., Schuller, M. G., Zelcer, A. A., Kirss, T. A.,Gore, S. S., and Germain M. J. (1977). Identificationand comparative analysis of thymidine phosphorylasein the plasma of healthy subjects and cancer patients.Journal of the National Cancer Institute, 58, 1587-1590.

Perper, R. J., Zee, T. W., and Mickelson, M. M. (1968).Purification of lymphocytes and platelets by gradientcentrifugation. Journal of Laboratory and ClinicalMedicine, 72, 842-848.

Phillips, D. R., and Morrison, M. (1970). The arrange-ment of proteins in the human erythrocyte membrane.Biochemical and Biophysical Research Communications,40, 284-289.

Sherlock, S. (1975). Diseases of the Liver and BiliarySystem, 5th edn., Blackwell: Oxford.

Straus, B., and Berenyi, M. R. (1973). Infection and im-munity in alcholic cirrhosis. Mt Sinai Journal ofMedicine NY, 40, 631-640.

Straus, B., Berenyi, M. R., Huang, J. M., and Straus, E.(1971). Delayed hypersensitivity in alcoholic cirrhosis.American Journal of Digestive Diseases, 16, 509-516.

Thestrup-Pedersen, K., Ladefoged, K., and Anderson, P.(1976). Lymphocyte transformation test with liver-specific protein and phytohaemagglutinin in patientswith liver disease. Clinical and Experimental Im-munology, 24, 1-8.

Thomas, D. W., Roberts, W. K., and Talmage, D. W.(1975). Regulation of the immune response: productionof a soluble suppressor by immune spleen cells in vitro.Journal of Immunology, 114, 1616-1622.

Wands, J. R., Perrotto, J. L., Alpert, E., and Isselbacher,K. J. (1975). Cell-mediated immunity in acute andchronic hepatitis. Journal of Clinical Investigation, 55,921-929.

Young, G. P., Van der Weyden, M. B., Rose, 1. S., andDudley, F. J. (1979). Lymphopenia and lymphocytetransformation in alcoholics. Experientia, 35, 268-269.

Zinneman, H. H. (1975). Autoimmune phenomena inalcoholic cirrhosis. American Journal of DigestiveDiseases, 20, 337-345.

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 G. P. Young, F. J. Dudley and M. B. Van Der Weyden transformationdisease sera on lymphocyte Suppressive effect of alcoholic liver

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