Awadalla, H. N.

Bedwany, R.

Brock, J.

Fahmy, M. H.

Ghanem, M. H.

Khalil, T.

Mucke, D.

Nofal, M.

S. Júnior, Helmy Khalil

Said, M.

Sawy, M. El

Youssef, M.

English

Cadernos Espinosanos (E-Journal)

Rev. Inst. Med. trop. São Paulo23(3) :97-I01, maío-junho, 1.981STUDY OF CELL MEDIATED IMMUNE RESPONSIVENESS TO SOI.UBLE EGGANTIGEN AND PHYTOHAEMAGGLUTININ USING THE LYMPHOBLASTTRANSFORMATION TEST IN HUMAN SCHISTOSOMIAS¡S MANSONI IN EGYPTHELntrY KHALIL S. JR,, M. H. FAHntrY, M. H, GHANEM, (Late), M. SAID, M. EL sAwY, M, NOFAL,H. N. AWADALLA, M. YOUSSEF, D, MUCKE, J. BROCK, R. BEDWANY and T. KHALILSUMMARYSoluble egg antigen and phytohaemagglutinin have been used to assess the cellmediated immune responsiveness in patients with schistosomiasis mansoni usingthe lymphoblast transformâtion test. soluble egg antigen gave a higher responsein cases 'with hepatosplenomegaly \Mhen compared with cases of simple intestinalbilharziasis, with a peak response at the youngest age groups. phytohaemaggluti-nin gave the lowest response in cases with simple intestinal bilharziasis and thehigher response was given by the middle age group. This supports the view ofthe importance of cell mediated immune state of the host in the various mani-festations of bilharziasis.The pathogenesis of schistosomal lesions isnow assumed to be parily due to both cell me_diated and humoral immunity ('WARREN r3).The bilharzial granuloma, the main etiolo_gical factor in the production of this disease isconsidered to be a manifestation of delayedn-ypersensitivity (W,{RREN et al. 1a).In a previous study the delayed intradermaltest and migration inhibition test showed gre_ater cell mediated immune responsiveness inpatients with bilharzial hepatic fibrosis (BHF)than those 'with simple intestinal bilharziasissuggesting a relationship between the immunestate of the patient and the clinicopathologi_cal picture of the disease (HELMY KHALIL etal. ó).In the present work, the lymphoblast trans_formation test (LTT), an in vitro correlative ofUDC 616.995 .r2ZI NTROD UCT I ONcell mediated immunity (CMI), has been usedto assess the delayed hypersensitivity in patients with bilharzial hepatosplenomegaly, andsimple intestinal bilharziasis using soluble eggantigen (SEA) as the specific antigen, and auniversal mitogen phytohaemagglutinin (pH.A) .MATERIAL AND METHODSSixty male bilharzial patients were subjecr"-ed to routine laboratory examinations whichincluded complete blood picture, stool examina-tion, plain X-ray chest, liver function tests andpercutaneous liver biopsy. Accordingly theyv¡ere subdivided as follows:Forty patients with BHF (Group I) as as_certained by liver biopsy which demonstratedthe presence of bilharzial ova, their remnanrsand the bilharzial pigments as well as the as.sociated pathological changes.Alexandria University, EgyptThis study was kindly sponsored. by the office of Nâval Research, \ryashington u.s.A., und.er contract wooo 14-24-c-0389; as well as by the scientific Research Academy, cairo Eglpt under project of rmmunology of BHFAuthors's address: Dr. samir Helmy Assaad Khalil. Ðepafiment of rnternal Med.icine. Faculty of Med.icine - .A,lexan-dria - Egypt97KHALIL S. JR., H.; FAHMY, M. H.; GHANEM, M. H.; SAID, M.; EL SAWY, M.; NOFAL, M.; AWADALLA, H. N.;YOUSSEF, M.; IUUCKE, D.; BROCK, J.; BEDWANY, R. & KHALIL, T. - Study of cell mediated immune respon-siveness to soluble egg antigen and phytohaemagglutinin using the iymphobl.ast transformation test in human schisto-somiasis mansoni ln Egypt. Rev. Inst. Med. troB. São Paulo 23:9?-101, 1981.Twenly patients with simple intest^nal biIharziasis (Group II), with dysentery as themain complaint. Stool examination showed liv-ing bilharzial ova, where liver biopsy did notshow hepâtic involvement.The study also included 40 male subjectsserving as controls and these \¡/ere further sub-divided âs follows:Fourteen normal healthy controls (GroupIIIa).Six controls with parasitosis other thanbilharziasis (Group IIIb) being infected witheither ascariasis, ankylostomiasis or enterobia-sis.Twenty controls with liver diseases otherthan BHF (Group IV).The latter two groups (III and IV) havebeen added to assess the specificity of the an-tigen; and they have been also subjected to thesame battery of investigations including liverbiopsy.Antigen PreparaúionThe SEA was prepared according to themethod of BOROS d¿ WARREN 2.Cell CultureThe method was essentially that of MAINIet aI.8 and \MAITHE & HIRSCHHORN 12. Thir-ty milliliters of heparinized blood were allow-ed to sediment at 37'C until the supernatant wassubstantially clear of RBCs. The supernatantu'hite blood cells rich fraction'was removed andcentrifuged at 1259 for ten minutes and the cellpellet was washed three times and resuspendedin Eagle's tissue culture medium suplementedwith HEPES 'ouffer (Hopkins & Williams 20mM,u100 ml), penicillin (100 units/ml), strepto-mycin (109 pglmD, L-glutamine (Ferek, FRG30 mg/100. mI) and 10% inactivated human ABserum. The culture tubes were set up in qua-druplets with various doses of SEA, withoutantigen and r¡/ith the non-specific mitogen PHA(lMelcorne), at a final dilution of. I%. The cul-tures were incubated at 3?'C in a humid at-modlitrère. They were the pulsed with one pCiper tube ôf 3H-thymidÍne (Amersham 28 Ci,/mMO) at the fourth day for 24 hours.9BìCell Harvesting and CountingThe cells were collected from the culturewells on the fifth day on-to glass fibres filters,dried at 60'C, washed successively with saline,57o triclrloroacetic acid (Prolabo) then coldmethanol (BDH). The samples were countedafter addition of 10 ml of scintillation flora(New England Nuclear Boston).The readings in counts per minute (cpm)have been converted into desintegration per mi-nute (dpm) from an efficiency curve plottedby means of standards (Packard). The degreeof response wes expressed in net dpm; calculat-ed as dpm of stimulated culture - dpm ofunstimulated culture.RESULTSPilot studies on the kinetics of the responseto SEA demonstrated that a peak response oc-curred after about 96 hours of culture and de-creased thereafter. Similarly dose response stu-dy showed that 10 pg of antigen containing pro-tein per culture resulted in maximum lympho-cyte transformation. Hence the peak was readafter 96 hours using 10 pg of antigen.A significant increase of increment blasttransformation was noticed in patients of groupI when compared with those of group II. Alsoa significant difference between group II andsubgroup fIIa v¡as noted (Tables Ia and b).TÀBLE I-AMean increment (net desintegration per minute) using SEA,in the diffetent groups studied in the lymphoblasttransformation test (LTT)Groutr)MeanÞ.u.104216.20 70212.55 258.50 259.29 256.67 310.808912.30 25862.80 102.34 102.16 112.53 25L.78Groups comparedGroup l/subgroup IIIaG¡oup ll/Subgroup IIIaSubgroup lllb/Subgroup IIIaGroup lvlsubgroup IIIaGroup l/Group IIT.ABLE I-BIntergroup significance usingIIIa IIIbWhen using 1%of increment blast43.3?10.0?30.0510.65337.52L2PHA, a significant increasetransformation was found< 0.001< 0.001> 0.05> 0.05< 0.001KHALIL S. JR., H.;.FAHMY,. M. H.i GHANEM, M. H.; SAID, M.; EL SA1ryy, M.; NOFAL,..M.; AWADATLA, ¡1. N.;YOUSSEF, lvl; MUCKE, D.; BR'OCK, J.; BEDIryANY, R,. & KH.{LIL, T. - Study of cell mediated immune respon-siveness to soluble egg antigen and phytohaemagglutinin using the lymphobtast transformâtion test in human schisto-somiasis mansoni in Egypt. Rev, Inst. Med. trop. São paulo ZB:92-101, 1991.in group I as compared to groups II and IIIa.No significant difference 'was seen betweensubgroup IIIa, IIIb and group IV (Tables IIaand b).TABLE II-A.Mean increment (d.p.m.) using l% pHA in the differentgroups studied in the LTTGroupMean 59898.95 4?379.10 5432,1-48 58005.1? 468?8.00s.D. 8483.87 5603.08 ?550.34 3326.51 10254.14Groups compaÌedTABLE II.BIntergroup significance using 10/o pHAGroup l/Subgroup IIIaGroup lllsubgroup IIIaSubgroup lllb/Subgroup IIIaGroup lvlsubgroup IIfaGroup l/croup II\ffhen LTT response to LVo pHA expressedas increment value was studied in relation toage, the middle age group (25-45 years) showedthe highest response in both groups I. and II(Tables IVa and b).TABLTI IV..\Mean increment (d.p.m.) of LTT using lolo pHA in thedifferent age groupsIIIb'When LTT in response to SEA was studi-ed as increment in relation to age, the bestresponse was observed in the youngest age ingroups I and II (Tables IIIa and b).TABLE III.AMean increment (d.p.m.) of LTT using SEA in the differenrage groupsIVAge groups2.1't23.0841.13317.845',15.96?8Groups:I.ê.ge groupsGroups:I< 0.05< 0.005> 0.05> 0.05< 0.001Means.D.< 25 years 25-45 years > 45 yearsMean 46600.63s.D. 3630.22MeanS.D.< 25 years 25-45 years > 45 years60675.548039.10Means.D.Means.D.Groups Age groups <25 years/Z'-41 years 25-45-years/>45 years110551.00 102993.?8 90658.006943.63 691?.86 ?63?.57T.â.BLE IV.BIntergtoup significance using 1% pHAMeanS.D.62497.67 42432.155347.96 2509.64IIIIII56827.51 51\24.20 46526,.6't4855.00 4342.89 17442.60Groups Age groups <25 years/25-45 years ZS-4S-yeârs/>45 yeats84795.005850.40TABLE III-BIntergroup significance using SEÁ50495.60 34910.502755.52 4?96.31ItIItIII ttpttp22r.86124.1571155.40 ?168.5017667.75 839.34DISCUSSIONThe sensitivit¡l of the LTT in relation roSEA is clearly demonstrated by the highty sig_nificant blastogenic response in bilharzial pa-tients as compared to healthy controls and. theother control groups. ,{ similar conclusion hasbeen be reached by COLLEy 3, WEISS et al. rsand I(Y et a1.7, in experimental animals and. inhuman patients. ABOUL-ENEIN et aI. r andWEISS et al. rs, when comparing the responseof LTT to SEA and, to worm antigen, obtainedsignificantly higher results with the former an_tigen.The antigenic specificity of LTT stimula_tion was exhibited by the inability of SEA rostimulate lymphocytes of normal control sub_jects in culture at the same concentration andalso by its inability to stimulate culture of lim_phocytes fro.m patients with other liver diseasesor control cases with other parasitosis. Theabsence of influence by other helminthic infec_99269.90 339.3385-44 176.240.8169> 0.052.5979< 0.020.1321> 0.053.r44r< 0.0052.0830> 0.050.9492> 0.057.2',t43< 0.00ro.oÐ ¡J< 0.0012.5700< 0.053.2492< 0.0057.0661< 0.00i1.748'l> 0.05KHALIL S. JR., H.; F.AHMY, M. H.; GHANEM, M. H.; SAID, M.; EL SA\ryY, M.; NOFAL, M.; AW.{DALLA, H. N.;YOUSSEF, M.; MUCKE, D.; BR'OCK, J.; BEDWANY, R. & KHALIL, T. - Study of cell mediâted immune respon.siveness to soluble egg antigen and phytohaemagglutinin using the lymphoblast transformation test in human schisto-somiasis mansoni in Egypt. Rev. Inst. Med. trop. São Paulo 23:9?-101, 1981.tions is in accordance with the finding ofSHERIF 10 when using extract of miracidia ofSchistosoma mansoni.Patients with bilharzial hepatosplenomega-ly showed a more significant response to SEAthan those with simple intestinal loilharziasis.However. COLLEY 4 stated that chronic infec-tion with S. mansoni led to diminished lympho-cyte transformation responsiveness to SEA. Ap-parently this is applicable to advanced cases.ABOUL-ENEIN et al. 1 also found increase inLTT response in cases with hepatomegaly yet,in the more severe cases with ascites, a dimini-shed response to SEA was found. None of ourpatients had shrunken liver or ascites.The response to PHA was somewhat higherin cases 'with hepatosplenomegaly when com-pared with controls and intestinal cases. Onthe other hand, ABOUL-ENEIN et aI. I foundno appreciable change in LTT response in bi-tharzial patients when compared with controlsexcept in advanced cases with ascites when de-pression was noticed and attributed that to se-condary immunodeficiency. Our results maysupport the view of an innate variation in cellmediated immunity which would be responsi-ble, amongst other factors, for the clinico-patho-logical outcome of the disease. The presentfindings are in accordance even with workersusing other models than schistosomiasis asTfI/ILKINSON ,& WHITE t0, who suggested a di-rect correlation between the state of delayedhypersensitivity response of the host and theextent of granuloma formation in tuberculosis.As regards the age, using SEA, the LTTshowed a greater response in those aged lessthan 25 years. SMITH et al. 11 related the floridreactions oloserved in young patients infectedwith S. haematobium, and the minimal tissueresponse in patients around 40 years, to schis-tosome egg production as well. as to host im-munological reactivity.Lymphoblastic transformation in responseto PHA gave a different age rqlation response,which was maximal in the middle age groupand agreed with the findingsof HAGEN &.FnØ-LUND s that a low response is elicited in veryyoung and very old people. Diminished respon'se in older age is due to involution of the thy-mus, though known to start before puberty,100yet the gland's epithelial cells continue to pro-duce thymosin and other hormones necessaryfor well functioning immune systems till the latetwenties when they gradually become deficient(MCBR,IDE 9).Thus, the higher LTT response observedin relation to SEA in patients with hepaticaffection and especially those aged less thantwenty five years, in conjunction with the re-sults obtained with PHA, points to a possiblerelationship between the host cell mediated im-munity and the different clinico-pathologicalmanifestations of bilharziasis.RESUMOEstudo da resposta imunológica celular a an-tígeno solúvel de ovo e fitohemaglutinina atra.vés do teste de transformação linfoblástica naesquistossomose mansônica humana no EgitoAntígeno solúvel de ovo e fitohemaglutini-na foram usados para medir a resposta imu-nológica celular dos pacientes com esquistosso-mose mansônica através do teste de transfor-mação linfoblástica. Antígeno solúvel de ovomostrou respostâ maior nos casos com hepa-tosplenomegalia quando comparados com asformas intestinais simples, as respostas maisaltas sendo reencontradas nos grupos etáriosmais jovens. Fitohemaglutinina mostrou respos-ta menor nos casos de esquistossomose intesti-nal simples e resposta maior nos grupos etáriosde média idade. Esses resultados corroborarna importância da resposta imunológica de tipocelular do hospedeiro nas várias manifestaçõesda esquistossomose.REFEBENCES1. ABOUL-ENEIN. M.; ABDEL KHALII<, A; NOOMAN,z.; SOLIMAN, A. & AHMED, T. - Studies of cellmediated immune reactions in schistosomiasis. EgyptiânJ. Haematol. 2: 7'l-L00, 79'16.2. BOROS, D. L. & WARREN, K' S - Delâyed hvpersen-sitivity type granuloma formation and dermal leactioninduced and elicited by a soluble factor isolated fromSchisúosoma mansoni eggs. Exper. Med. 132: 488-507,19?0.3. COLLEY, D. G. - SEA induced lymphocyte blastogene-sis in experimental mutine S. mansoni infection. Im'munology 10?: 14??-1480, 1971.KHALIL S. JR., H.; FAHMY, M. H.; GHANEM, M. H.; SAID, M.; EL SAWY, M.; NOFAL, M.; AWADALLA, H. N.;YOUSSEF, M.; MüCKE, D.; BROCK, J.; BED\{/ANY, R. & KHALIL, T. - Study of cell mediated immune respon-siveness to soluble egg antigen and phytohaemagglutÍnin using the lymphoblast transformation test in human schisto-somiasis mansoni in Egypt. Rev. Inst. Med. trop. São Paulo 23:97-101, 1981.COLLEY, D. G. - The immunopathology of schisto-somiasis. ln: Recent Advances in Clinical Immunology.R,. A. Thompson (Editor). London, Churchil.l Livings-tone, 1977, 101-122.HAGEN, C. & FR,ØLUND, Â. - Anâlysis in the varia.tion of lymphocyte response to PHA in normal sub-jects. Acta Path. Mlcrobiol. Scand. 8l: 253-258, 19?3.HELMY KHALIL JR., S.; FAHMY, M. H.; GHANEM,M. H.; SAID, M.; EL-SAWY, M.; NOFAL, M.; ¿.WA-DALLA, H. N.; YOUSSEF, M.; MUCKE, D. & BROCK,J. - Cell mediated immune (CMI) responsiveness tosoluble egg antigen (SEA) and its relation to theoccuIrence of schistosomal hepatosplenic disease in pa-tients with schistosomiasis mansoni. TroDenmed. Pa.rasit. 30: 426-428, 1919.KY, N.; HALPERN, B. & ROCHE, L. - Lymphocyteculture in the immunological diagnosis of human bi-lhaÌziasis. Ann. AIIergy 38: 353-356, 19?7.MAINI, R,. N.; ROFFE, L. M.; MAGRATIÍ, I. T. &ÐURMONDE, D. C. - Standardization of leucocyte mi-gration test. Inúern. Arch. Allergy 45: 308-321, 1969.McBRIDE, G. - Thymus gland attains new staturein age of immunopharmacology. J.A.M.A. ZBj: 429-488,1977.SHERIF, .{. F. - A new intradermal antigen for thediagnosis of schistosomiasis. Ann. Trop. Med. para.sitol. 50: 105.112, 1956.II SMITH, J. H.; KAMEL, L A.; ELWI, A. & von LICH-TENBERG, F. A. - A quantitative post-mortern ana-lysis of urinary schistosomiasis in Egypt. II. Evolu-tion and epidemiology. Am. J. Trop. Med. Hyg. 24:806-822, 19?5.WAITHE, r.V. I. & HIRSCHHORN, K. - The lymphocy.te response to activators. In: Handbook of ExDerimen.tal Imunology. Part II. Cellular Immunology. Ox-ford, Blackwell Scientific Publications 25.1 - 25.10,r9?3.WARREN, K. S. - The immunopathogenesis of schis-tosomiasis: A multidisciplinary approach. Trans. Roy.Soc. Trop. Med, Hyg. 66: 417-432, 79'12.WAR'REN, K. S.; DOMINGO, E. O. & CO!,VAN, R. B.- Granuloma formátion around Schistosoma eggs as amanifestation of delayed hypersensitivity. Am. J. Path.51: ?35-756, 196?.WEISS, N.; OBERLIN, U. P. & DEGRÉMONT - Sti-mulation of hamster and human lymphocyte culturesby soluble egg antigens (SEA) of Schisúosom¿ haema-tobium and S. mansoni, Trans. Roy. Soc. Trop. Med.Hyg. ?0: 37742f, 7916.WILKINSON, P. C. E¿ WHITE, R. G. - The IoIe ofmycobacteria and silica in the immunological responseof guinea pig. Imunology 11: 229-241, 1966.12.l4t6Receþido para puþlicação em Z7/5/L9B}.101

Study of cell mediated immune responsiveness to soluble egg antigen and phytohaemagglutinin using the lymphoblast transformation test in human schistosomiasis mansoni in Egypt

https://www.revistas.usp.br/rimtsp/article/download/197052/181400

Study of cell mediated immune responsiveness to soluble egg antigen and phytohaemagglutinin using the lymphoblast transformation test in human schistosomiasis mansoni in Egypt

Abstract

Similar works

Full text

Available Versions

Cadernos Espinosanos (E-Journal)