Effect of camel milk on thymus and activation-regulated chemokine in autistic children: double-blind study

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1 nture publishing group Clinicl Investigtion Effect of cmel milk on thymus nd ctivtion-regulted chemokine in utistic children: double-blind study Shhid Bshir 1,2 nd Lil Y. Al-Aydhi 1 Bckground: This study imed to investigte the role of the effectiveness of cmel milk (CM) (rw nd boiled) on thymus nd ctivtion-regulted chemokine (TARC) serum levels nd childhood utism rting scle (CARS) score in subjects with utism nd compred to plcebo group (cow milk). Methods: Forty-five subjects dignosed with utism were rndomly ssigned to receive boiled CM for group I (n = 15), rw CM for group II (n = 15), nd plcebo for group III (n = 15) for 2 wk. Mesures included chnges in professionlly completed CARS score nd blood smples for TARC serum level were tken before nd fter milk consumption of 500 ml per dy in children s regulr dily diet. Results: The serum levels of TARC decresed significntly (P = 0.004) in boiled CM nd in rw CM group (P = 0.01) too, but no effect ws observed (P = 0.68) in plcebo group. Furthermore, significnt improvements were observed in CARS score (P = 0.04) in rw CM group only. There were no significnt reltionships between the serum of TARC level nd the CARS score, ge, or gender for ny group. Conclusion: CM dministered for 2 wk significntly improved clinicl mesurements of utism severity nd decresed serum level of TARC in utistic children, but subsequent studies re recommended. Autism is severe neurodevelopmentl disorder tht is chrcterized by impirment in verbl nd nonverbl communiction, imgintion, reciprocl socil interction, nd evidence of developmentl dely within the first 3 y of life (1 4). Immunologicl nd environmentl fctors, such s diet, infection, nd xenobiotics ply criticl roles in the development of utism (4 6). Over the yers, reserch findings, especilly from our lb, suggested possible involvement of ltered immune system in the pthophysiology of utism spectrum disorder (ASD) (7,8). Despite the cler unmet medicl need, currently, there is no recognized effective comprehensive tretment (9). Proinflmmtory chemokines, such s monocyte chemotctic protein-1 nd thymus nd ctivtion-regulted chemokine (TARC), long with cytokines, such s tumor necrosis fctor α, were consistently elevted in the brins of individuls with utism (7,8). At criticl times of infntile development, immune dysregultion my result in the relese of immunemodultory molecules, such s chemokines nd cytokines, leding to ltered neuronl development nd neurl function (10,11). Furthermore, Ashwood nd collegues (2008) found tht reduced levels of the modultory cytokine, trnsforming growth fctor-β1 (TGF-β1), in utistic children contributed to the dysregultion of dptive behviors nd predisposl for utoimmune responses (12). Milk is n importnt nutrient in humn nourishment. In some communities, cmels represent the most importnt source of this nutrient. Cmel milk (CM) hs emerged to hve potentil therpeutic effects in diseses such s dibetes (13,14), heptitis B (15), possibly certin symptoms ccompnying utism enterocolitis, H. pylori infection nd lctse deficiency might be cured with CM s well (16). Recently, some prents hve been using cmels milk s tretment in some children with ASD becuse cmels milk ppers to help food llergies in some individuls (17,18). CM s lctoferrin hs very high levels of bctericidl nd bcteriosttic properties ginst Grm-positive nd Grm-negtive bcteri (19), more thn cow nd humn lctoferrin. CM contins vrious protective proteins, minly enzymes which exert ntibcteril nd immunologicl properties (20). The fct tht CM lcks β-lctoglobulin nd new β-csein (21), two powerful llergens in cow milk, mkes the milk ttrctive for children suffering from milk llergies (22). Phylogenetic differences could be responsible for the filed recognition of cmels proteins by circulting IgEs nd monoclonl ntibodies (23). Children with severe food llergies improved rpidly with CM. CM with its unique properties, such s, high vitmin C levels, low ft content, nd low moleculr weight immunoglobulins, mkes n idel nturl intervention method in ASD. The hypothesis tested in the present study ws tht TARC ct on their chemokine receptor type 4 (CCR4) receptors to enhnce the recruitment nd ctivtion of T helper 2 cells with subsequent production of type 2 cytokines tht include interleukin (IL)-4, IL-5, IL-9, nd IL-13 (24,25). CCR4 lignds hve n importnt pthogenic role in inflmmtory conditions such 1 Deprtment of Physiology, Autism Reserch nd Tretment Center, Shik AL-Amodi Autism Reserch Chir, Fculty of Medicine, King Sud University, Riydh, Sudi Arbi; 2 Division of Cognitive Neurology, Deprtment of Neurology, Berenson-Allen Center for Noninvsive Brin Stimultion, Beth Isrel Deconess Medicl Center, Hrvrd Medicl School, Boston, Msschusetts. Correspondence: Lil Y. Al-Aydhi (ydh2@gmil.com) Received 19 Februry 2013; ccepted 14 September 2013; dvnce online publiction 29 Jnury doi: /pr Copyright 2014 Interntionl Peditric Reserch Foundtion, Inc. Volume 75 Number 4 April 2014 Peditric Reserch 559

2 Bshir nd Al-Aydhi s llergy nd some utoimmune diseses (26 28). TARC nd their receptors hve been implicted s functionl meditors of immunopthology of utoimmune neuroinflmmtory diseses (29,30) nd childhood utism rting scle (CARS) score which hve significnt impct on behvior, cognition, sociliztion, nd helth/physicl trits ssocited with n ASD dignosis. The present prospective, double-blind, plcebo-controlled tril evluted whether stndrdized CM dministered to ptients dignosed with n ASD on dily bsis for 2 wk would result in improved CARS score trits ssocited with n ASD dignosis. RESULTS The generl chrcteristics of the study prticipnts nd the results of the serum levels of TARC re depicted in Tble 1. No significnt differences were observed between the CM (rw or boiled) nd plcebo groups with respect to ge, gender, or ASD dignostic sttus. There were no significnt differences on serum levels of TARC t bseline between the rndomiztion groups (P = 0.28) nd on the CARS score between groups (P = 0.719). Figure 1 summrizes the chnge in serum levels of TARC fter the CM (rw or boiled) nd plcebo groups following 2 wk of therpy. Chnges in serum levels of TARC significntly decresed (P = 0.004, P = 0.01, for vlues see Tble 2) for CM (boiled nd rw) group respectively. In contrst, no similr chnges were observed (P = 0.54, for vlues see Tble 2) for Tble 1. Bseline chrcteristics of cmel milk group (rw nd boiled) nd plcebo (cow milk) cohorts t rndomiztion Cmel milk group Rw Boiled Plcebo group Age (yer) 7.1 ± ± ± 4.3 Gender (n) Mle Femle Men ± SD. Men TARC vlue (pg/ml) Rw cmel milk group Boiled cmel milk group Plcebo group Figure 1. Serum levels of thymus nd ctivtion-regulted chemokine (TARC) before (blck columns) nd fter (white columns) in cmel milk group (rw nd boiled) nd plcebo (cow milk) group of utistic children. plcebo group. There ws significnt difference (P = 0.04, for vlues see Tble 3) for CARS score from ± 5.3 (men ± SEM) to 33.8 ± 2.7 (men ± SEM) in rw CM group only. No significnt correltion ws found between serum TARC levels nd CARS score for children with ASD for ny group. DISCUSSION The present study is the first prospective, double-blind, plcebo-controlled tril to evlute the effects of CM therpy mong subjects dignosed with n ASD. In the present study, CM therpy (rw CM group) for 2 wk mong subjects dignosed with n ASD significntly improved clinicl mesurements (CARS score) recorded by trined professionl nd prents of study subjects. Furthermore, CM therpy (rw nd boiled) significntly decresed serum levels of TARC mong ptients dignosed with n ASD. Finlly, CM therpy ws generlly well tolerted with miniml dverse effects. The side effects in the children who did not tolerte the tretment well were irritbility nd/or stomch discomfort. CM, with its distinctive properties, could be promising beneficil intervention pproch in ASD. Mmmlin ntibodies composed of two identicl H-chins nd two identicl L-chins (31). Cmel IgG ntibodies re hevy-chin ntibodies tht lck the L-chin (31). The size of the ntibodies is mjor issue in the development of humn immunotherpy. Cmel ntibodies re only one tenth of the size of humn ntibodies, which mkes them nturl nno-bodies (31,32). Furthermore, the high content of vitmin C in CM gives it string ntioxidnt property (33). Recent reports hve demonstrted higher oxidtive stress sttus in ASD subjects compred to normlly developing controls (34 36), which mkes CM n idel ntioxidnt food for ASD subjects. In ddition, those unique properties of CM most probbly reduced TARC synthesis nd secretion, nd consequently, reducing the neuroimflmmtion nd the utoimmune rection, leding to improved behvior, reflected on improved CARS scoring results. Tble 2. Serum levels of TARC results for cmel milk group (rw, boiled) nd plcebo (cow milk) groups Before TARC (pg/ml) After Boiled cmel milk group (n = 15) ± ± 3.08* Rw cmel milk group (n = 15) ± ± 5.9* Plcebo group (n = 15) ± ± 11 TARC, thymus ctivtion-regulted chemokine. Men ± SD. *Significnce level (P = 0.05). Tble 3. Childhood utism rting scle scoring results for cmel milk (rw, boiled) nd plcebo (cow milk) groups Before After Boiled cmel milk group (n = 15) 38 ± ± 2.7 Rw cmel milk group (n = 15) 37 ± ± 2.6* Plcebo group (n = 15) 36 ± 3 33 ± 3.4 Men ± SD. *Significnce level (P = 0.05). 560 Peditric Reserch Volume 75 Number 4 April 2014 Copyright 2014 Interntionl Peditric Reserch Foundtion, Inc.

3 Chemokines nd utism However, cler pttern hs emerged over severl studies tht shows ltered levels of immune meditors re ssocited with incresed impirments in behviors (7,12,37,38) nd suggests tht dysregulted immune response is relted to behviorl nd cognitive impirments in children with ASD. The elements such s zinc, copper, selenium, nd iron re not likely to hve influenced our results, since their mounts in CM nd in cow milk re prcticlly the sme (39). Strengths/Limittions The min strength of the present study is the design s prospective, double-blind, plcebo-controlled tril. Every effort ws mde to ensure tht the present study ws truly doubleblind so tht those evluting study subjects, both trined professionls nd prents, hd no knowledge s to the tretment sttus of ny prticulr study subject. Furthermore, the present study lso ttempted to minimize the effects of study drop-out for potentil dverse rections in the dt, especilly for CARS scores. These prticulr scoring mesurements were conducted by the study investigtors on ech child regrdless of whether or not they dropped-out from the study for potentil dverse rections. For ech outcome mesurement evluted, the reltive chnge for the prmeter following 2 wk of therpy in comprison to bseline ws exmined. As result, potentil vrition between study subjects ws minimized becuse ech study subject served s his or her own control. One of the potentil limittions of the present study is the smll smple size exmined. The smll smple size in the present study my hve resulted in specific effects of CM therpy being missed becuse of lck of sttisticl power to detect significnt chnges between the CM (rw or boiled) nd plcebo groups. As result, the observtion of significnt positive effects of CM therpy in the present study tends to rgue tht the observed effects represent genuine phenomen. The dt from the present study provide the bsis for lrger, more focused study on the promising elements. A further potentil limittion of the present study is the exct mechnism of ction of CM ws not elucidted from the present study. Finlly, n dditionl potentil limittion of the present study is the fct tht the dose of CM used my not hve been optiml. The dosing regimen of CM used in the present study ws derived from peditric nutrition, s the recommended strting dose for children. Conclusion Cmels immune systems re stronger thn tht of humns nd the smll immunoglobulins pss from the CM into the humn blood. As immunoglobulins re found in CM throughout lcttion, drinking milk will provide tool for combting utoimmune diseses by rehbilitting the immune system rther thn is depression. In conclusion, the results of the present study suggest tht CM therpy over the course of 2 wk of therpy significntly improved clinicl mesurements of ASD severity (CARS score). Furthermore, there were significnt decrese levels of serum of TARC mong the study subjects exmined. Overll, the CM therpy ws well tolerted. It is suggested tht future studies further explore the biologicl bsis for CM s mode of ction t the cellulr level in those ptients dignosed with n ASD who would most benefit from CM therpy. METHODS Subjects A totl of 45 subjects dignosed with ASD, ged from 2 to 12 y (40 mles, 5 femles), were recruited to the study. The study subjects hve body weight between 12.8 kg nd 42.6 kg. None of the study subjects hd previously received CM therpy. None of the study subjects hd ny chnge in therpy or tretment (including medictions) within 1 mo prior to the study. Ptients fulfilled the criteri for the dignosis of utism ccording to the 4th edition of the Dignostic nd Sttisticl Mnul of Mentl Disorders (2). The study protocol received Institutionl Review Bord pprovl from King Khlid Hospitl (King Sud University, Riydh, Sudi Arbi). All prents signed consent form nd ll received copy. Clinicl Assessment Autism Dignostic Observtion Schedule is semistructured, stndrdized observtionl instrument to ssess the socil nd communictive bilities of individuls with possible ASD. Items re scored from 0 (not bnorml) to 2 or 3 (most bnorml), nd dignosis of utism or ASD is estblished if the individul ssessed hs scores higher thn the estblished cut-off vlues in the communiction domin, the socil domin, nd sum of the two (40). Clinicl Mesure Childhood utism rting scle. Study prticipnts were evluted using CARS test conducted only by single child psychitrist who observed the subjects nd interviewed the prent(s) nd ws unwre s to the tretment sttus of the subject. The CARS test is 15-item behviorl rting scle (relting to people, emotionl response, imittion, body use, object use, listening response, fer or nervousness, verbl communiction, nonverbl communiction, ctivity level, consistency of intellectul response, dpttion to chnge, tste, touch nd smell response, nd generl impressions) developed to identify utism s well s to quntittively describe the severity of the disorder. The CARS test is well-estblished mesure of utism severity (41). The internl consistency relibility lph coefficient is 0.94; the interrter relibility correltion coefficient is 0.71; nd the test-retest correltion coefficient is 0.88 (42). CARS scores hve high criterion-relted vlidity when compred to clinicl rtings during the sme dignostic sessions, with significnt correltion of 0.84 (42). Lb Testing Blood smples. After n overnight fsting, blood smples (3 ml) were collected from subjects in both groups in plin test tubes. Blood smples were llowed to clot nd then centrifuged t 3,000 rpm to collect serum smples, which were stored frozen in freezer t 80 C until the time of nlyticl ssys. The detil procedure hs been described in our previous work (7,8,43) Chemokine ssy. Serum level of TARC ws mesured using commercilly vilble sndwich enzyme immunossy (ELISA) kit from CUSABIO BIOTECH (Wuhn, Chin). Study Milk CM (boiled or rw) ws supplied in liquid preprtion. The plcebo (cow milk) group ws identicl in ppernce. The recommended childhood strting dose of 500 ml milk per dy (hlf the totl dose dministered in the morning nd hlf the totl dose dministered in the evening). Study subject specific dosing instructions were plced on ech liquid preprtion provided to study subjects. Prents were sked to continue with the children s dily routines. They were not llowed to dd or remove ny interventions such s diet plns, supplements, or phrmcotherpies throughout the study period. 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4 Bshir nd Al-Aydhi Milk hndling. Fresh CM ws obtined from trusted cmel frm tht rn regulr routine veterinry checkups on the cmels. After receiving the milk, microbiologicl screening tests were conducted on ll milk btches to ensure tht it ws free of pthogens commonly found in rw CM (44). The pthologicl screenings were conducted to detect Cmpylobcter (KGA, Drmstdt, Germny) Bcillus cereus enterotoxin, E. coli O157:H7, Listeri, Slmonell by GLISA rpid testing using the kits Singlepth Cmpylobcter, Duopth Cereus Enterotoxin (EMD chemicls), Revel E. coli O157:H7, Slmonell, Listeri (Neogen), nd B. Brucell (Anigen). Any btch-tested positive for the mentioned pthogens ws immeditely excluded from the study. CM supplied to group I ws psteurized by heting to 65 C for 15 s, then removed, cooled in ice pot initilly nd then stored in the freezer t 80 C. Milk supplied to group II ws not heted to void losing beneficil nutrients nd proteins (45). Frozen milk ws supplied to ptients using bisphenol-a-free freezer bottles nd thwed on countertops s needed. Study Design This ws rndomized, double-blind, plcebo-controlled study. The study ws conducted between 2011 nd The study subjects were recruited through community contcts. The study protocol clled for 36 subjects to receive CM (rw or boiled) nd 18 study subjects to receive plcebo (cow milk). A totl of 54 subjects were recruited for the present study. Four subjects withdrew prior to rndomiztion into CM or plcebo groups. A totl of 54 subjects were rndomly ssigned to receive CM (boiled or rw) or plcebo, nd of these, totl of 9 subjects (4 in boiled, 2 in rw in CM group nd 3 in the plcebo group) withdrew prior to successful completion of 2 wk of therpy. Among the nine subjects withdrwing from the study prior to successful completion of 2 wk of therpy, four subjects withdrew becuse of dverse rections (two in rw CM group, one in boiled CM group nd 1 in the plcebo group), three subjects did not comply with the study protocol, nd two ws lost to follow-up with no known dverse rection. In ddition, study investigtors monitored study subjects to ensure complince nd to monitor for potentil dverse rections. Prerndomiztion Phse Study subjects were seen for n initil screening where study investigtors obtined informtion regrding demogrphics, forml dignosis, ge t dignosis, ge of pprent onset, informtion regrding dely or regression, ny current medicl issues, medictions, bodyweight, nd llergies on ech study subject. A bseline CARS evlution ws performed by child psychitrist. In ddition, blood smples were collected on ech study subject t n utism reserch nd tretment center drw sttion. Rndomiztion Phse Following the initil screening nd collection of lbs, ll study subjects strted therpy within 30 d of bseline mesurements. A study investigtor, who did not perform ny clinicl mesurements on study subjects, used coin-flip to rndomly ssign study subjects to either the CM (rw or boiled) or plcebo groups. Since there ws difference in smple size between the CM nd plcebo groups, the plcebo group ws filled with study subjects before the tretment group, so tht the ltter study subjects were ll ssigned to the CM group (rw or boiled). Study investigtors in contct with the study subjects nd the prents of study subjects were not informed of the tretment sttus (CM/plcebo) of ech study prticipnt until ll study subjects hd completed the tril, nd hence the ssignment (CM/plcebo) strtegy used should not hve reveled ny informtion regrding the tretment sttus of ny study prticipnt to study investigtors in contct with study subjects nd the prents of study subjects. For the durtion of the tril, ny concomitnt use of drugs/supplements were not chnged s fr s possible. Sttisticl Anlysis The results were nlyzed using the commercilly vilble softwre pckge Sttview (Abcus concepts, Berkley, CA). The dt re presented s the mens ± SEM. The Mnn Whitney test ws used for comprisons between dt. The null hypothesis ws tht there would be no difference in the dt distributions of the reltive chnge in test results following 2 wk of tretment in comprison to bseline mesurements between study subjects receiving CM (rw or boiled) in comprison to plcebo (cow milk). In ddition, the reltionship between the chnge in serum levels of TARC following 2 wk of tretment in comprison to his or her bseline mesurements, nd the chnges in specific outcome mesurements (CARS scores) 2 wk of tretment in comprison to his or her bseline mesurements. Spermn s rnk correltion coefficient r ws used to determine the reltionship between vribles. 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