research article achalasia an autoimmune inflammatory...

Research ArticleAchalasiamdashAn Autoimmune Inflammatory DiseaseA Cross-Sectional Study

J Furuzawa-Carballeda1 D Aguilar-Leoacuten2 A Gamboa-Domiacutenguez2

M A Valdovinos3 C Nuntildeez-Aacutelvarez1 L A Martiacuten-del-Campo4 A B Enriacutequez1

E Coss-Adame3 A E Svarch4 A Flores-Naacutejera4 A Villa-Bantildeos4

J C Ceballos4 and G Torres-Villalobos4

1Department of Immunology and Rheumatology National Institute of Medical Sciences and Nutrition Vasco de Quiroga No 15Colonia Seccion XVI 14000 Mexico City DF Mexico2Department of Pathology National Institute of Medical Sciences and Nutrition Vasco de Quiroga No 15Colonia Seccion XVI 14000 Mexico City DF Mexico3Department of Gastroenterology National Institute of Medical Sciences and Nutrition Vasco de Quiroga No 15Colonia Seccion XVI 14000 Mexico City DF Mexico4Department of Experimental Surgery National Institute of Medical Sciences and Nutrition Vasco de Quiroga No 15Colonia Seccion XVI 14000 Mexico City DF Mexico

Correspondence should be addressed to G Torres-Villalobos torresvgmyahoocommx

Received 15 January 2015 Revised 24 March 2015 Accepted 14 April 2015

Academic Editor Ghislain Opdenakker

Copyright copy 2015 J Furuzawa-Carballeda et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Idiopathic achalasia is a disease of unknown etiology The loss of myenteric plexus associated with inflammatory infiltratesand autoantibodies support the hypothesis of an autoimmune mechanism Thirty-two patients diagnosed by high-resolutionmanometry with achalasia were included Twenty-six specimens from lower esophageal sphincter muscle were compared with5 esophagectomy biopsies (control) Immunohistochemical (biopsies) and flow cytometry (peripheral blood) analyses wereperformed Circulating anti-myenteric autoantibodies were evaluated by indirect immunofluorescence Herpes simplex virus-1 (HSV-1) infection was determined by in situ hybridization RT-PCR and immunohistochemistry Histopathological analysisshowed capillaritis (51) plexitis (23) nerve hypertrophy (16) venulitis (7) and fibrosis (3) Achalasia tissue exhibitedan increase in the expression of proteins involved in extracellular matrix turnover apoptosis proinflammatory and profibrogeniccytokines andTregs andBregs versus controls (119875 lt 0001) CirculatingTh22Th17Th2Th1percentage showed a significant increaseversus healthy donors (119875 lt 001) Type III achalasia patients exhibited the highest inflammatory response versus types I and IIPrevalence of both anti-myenteric antibodies and HSV-1 infection in achalasia patients was 100 versus 0 in controls Our resultssuggest that achalasia is a disease with an important local and systemic inflammatory autoimmune component associated with thepresence of specific anti-myenteric autoantibodies as well as HSV-1 infection

1 Introduction

Idiopathic achalasia is one of the most common causes ofesophageal motor disorders Manometrically it is charac-terized by aperistalsis failure of lower esophageal sphincter(LES) relaxation in response to swallows and increased LESresting tone Clinical manifestations include dysphagia to

solids and liquids but the patients may also have regurgi-tation of esophageal contents chest pain cough aspirationweight loss and heartburn [1 2] Achalasia affects both sexesand all age groups The prevalence of the disease is eightcases per million population with an estimate incidence ofapproximately 03ndash163 per 100000 habitants per year [34] Diagnosis is established by high-resolution manometry

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2015 Article ID 729217 18 pageshttpdxdoiorg1011552015729217

2 Journal of Immunology Research

(HRM) as the gold standard while the esophagram withbarium swallow and endoscopy is useful to rule out otherpathologies Idiopathic achalasia has been classified in threetypes based on the HRM achalasia with minimal esophagealpressurization (type I) achalasia with esophageal compres-sion (type II) and achalasia with spasm (type III) [5]

Even though the disease was first described morethan 300 years ago etiology and physiopathology are stillunknown However many contemporary hypotheses suggesta multifactorial etiopathogenic mechanism [6 7] Thuspathophysiologically idiopathic achalasia is caused by loss ofinhibitory ganglion in the myenteric plexus of the esophagus[7ndash11] In order to explain this several studies have attemptedto explore initiating agents that may cause the disease suchas latent or active viral infection Proposal virus candidateshave included herpes simplex virus (HSV) (neurotropic viruswith a predilection for squamous epithelium) bornavirusvaricella zoster (VZ) measles and human papilloma virus(HPV) [12 13] Nonetheless not all the patients with viralinfections develop the disease Numerous potential genetictargets exist in the immune system which may createsusceptibility Some studies have been shown genetic cor-relations in achalasia for example ALADIN gen associ-ated with Allgrove syndrome receptor of vasoactive intesti-nal polypeptide (VIPR1) gene polymorphism interleukin-(IL-) 23 receptor polymorphism protein tyrosine phos-phatase nonreceptor 22 gene polymorphism and humanleukocyte antigen- (HLA-) DQ1205731 (119867119871119860-1198631198761198611lowast0503 and119867119871119860-1198631198761198611lowast0601) HLA-DQ1205721 (119867119871119860-1198631198761198601lowast0103) andHLA-DQ1205731 (119867119871119860-1198631198761198611lowast0301 and119867119871119860-1198631198761198611lowast0304) [8]Consequently chronic viral infection could trigger aberrantimmune response under appropriate genetic and environ-mental background allowing the loss of esophageal neuronsMoreover detailed examination of Auerbachrsquos plexus hasshown infiltration of CD3+CD45RO+ T cells predominantlyCD8+ T cytotoxic lymphocytes expressing activationmarkers[7 10 11 14] Notwithstanding little is known about whetherthese subpopulations of cells are truly autoimmune as inthe case of pathogenic Th22 and Th17 effector cell subsetsand what happens with cellular mechanisms which areparticipating in an attempt to regulate the inflammationsuch as regulatory CD4+ T cells and IL-10-producing B cells[15 16] Finally in accordance with the hypotheses evidenceautoantibodies against myenteric neurons have been shownin serum samples from patients with achalasia especiallyin those with HLA DQA1lowast0103 and DQB1lowast0603 alleles[17] However no specific antigenicity of myenteric neuronautoantibodies has been identified [2 18]

Based on these data and hypotheses the aim of the studywas to analyze in esophageal muscle tissue and peripheralblood the localized and systemic inflammation patterns inidiopathic achalasia patients Furthermore we evaluate theexistence of circulating anti-myenteric autoantibodies andtheir specificities in sera frompatients and assess the presenceof herpes simplex virus type 1 (HSV-1) in esophageal muscle

2 Materials and Methods

21 Study Design This was an exploratory observationaland cross-sectional study designed to identify by the first

time the presence of CD4+ T-cell subpopulations relatedto autoimmune diseases autoantibodies and their speci-ficity and proteins involved in extracellular matrix turnoverapoptosis proinflammatory and profibrogenic cytokinesand regulatory T and B cells (Tregs and Bregs) in loweresophageal sphincter biopsies from achalasia patientsThirty-two patients with idiopathic achalasia without Chagas dis-ease HIV or HCV infections were included between January2013 and July 2014 The medical records of each patientwere reviewed Peripheral blood was obtained from 20 age-matched healthy donors (HD) who volunteered at the bloodbank HDs were also interviewed in order to discard any dis-ease including known autoimmune disease use of immuno-suppressors or prednisone and concurrent infections Fivebiopsies of esophagectomy from patients with squamous cellcarcinoma from upper third of esophagus were included ascontrol tissue samples (see Supplementary Figure 1 in Supple-mentaryMaterial available online at httpdxdoiorg1011552015729217)

22HighResolution EsophagealManometry Protocol Ahigh-resolution esophageal manometry was performed in everypatient at baseline and before being referred to surgeryA solid-state HRM probe with 36 circumferential sensorswas used (Given Imaging Yokneam Israel) Having thepatient in a sitting position and at 45 degrees stationaryHMR was performed After a 12 hr fasting period the probewas inserted transnasally until passing the esophagogastricjunction assessed visually in the computer screen Ten waterswallows of 5mL separated by 30 seconds were provided

Analyses were performed using Manoview 20 (GivenImaging) and patients were classified according to latestChicago classification [19] into three groups (1) type I acha-lasia (without pressurization within the esophageal body)(2) type II (with pan-pressurization) and (3) type III (withspasm) Classification was performed by two gastroenterol-ogists (MAV E-CA) experts in high-resolution esophagealmanometry Surgeon was blinded to the high-resolutionmanometry analysis [19]

23 Erythrocyte Sedimentation Rate Erythrocyte sedimenta-tion rate (ESR) was determined by Westergren method

24 Immunohistochemistry Once the myotomy was done a2 cm long and 2mm width tissue was obtained Tissue wasimmediately formalin-fixed and paraffin embedded Four120583m thick tissue sections were deparaffinized and rehydratedTissues were incubated for 18 h at 4∘C with goat polyclonalanti-human MMP-9 TIMP-1 TGF-1205731 IL-22 IL-17A FASor mouse monoclonal IgG

1HSV-1 antibody (Santa Cruz

Biotechnology Santa Cruz CA USA) at 10120583gmL Bind-ing was detected by incubating sections with biotinylateddonkey anti-goat IgG antibody (ABC Staining System SantaCruz Biotechnology) Slides were incubated with horseradishperoxidase- (HRP-) streptavidin and 331015840-diaminobenzidine(DAB) (Sigma-Aldrich) and counterstained with hema-toxylin Negative control staining was performed with nor-mal human serumdiluted 1 100 instead of primary antibodyThe reactive blank was incubated with phosphate buffer

Journal of Immunology Research 3

saline-egg albumin (Sigma-Aldrich) instead of the primaryantibody Both controls excluded nonspecific staining orendogenous enzymatic activities At least two different sec-tions and two fields (times320) were examined for each biopsyImmunostained cells were assessed by estimating the numberof positively staining cells in two fields (times320) and werereported as the percentage of immunoreactive cells Resultsare expressed as themeanplusmn standard error of themean (SEM)of cells quantified by the program Image Pro Plus version 511[15]

25 Double-Staining Procedure Todetermine the subpopula-tion of IL-13+IL-4+- CD4+IFN-120574+-expressing T cellsCD25+Foxp3+ regulatory T cells and CD20+IL-10+-producing B-cell subpopulations a simultaneous detectionwas performed [EnVision G|2 Doublestain System (DakoGlostrup Denmark)] The procedure is a sequential doublestaining where the first antigen was visualized usingHRP3101584031015840-diaminobenzidine (DAB) and the second antigenwas visualized using alkaline phosphatase (AP)permanentred Briefly incubation of samples with 200 120583L of dualendogenous enzyme block was performed This procedureinhibited endogenous AP peroxidase and pseudoperoxidaseactivity present in tissues After blocking tissues wereincubated with 200120583L of normal serum as negative controlor second primary rabbit polyclonal anti-CD20 anti-CD25 IgG antibody or mouse monoclonal anti-CD4 oranti-IL-4 IgG

1antibody (Santa Cruz Biotechnology) at

10 120583gmL Then 200120583L of rabbitmouse LINK was added for10min Tissues were incubated with 200120583L of polymerAPreagent Reaction was visualized by incubation with 200 120583Lpermanent red chromogen Tissues were counterstained withhematoxylin and mounted in aqueous mounting mediumAt least two different sections and two fields (times320) wereexamined for each biopsy Double positive IL-4+IL-13+-CD4+IFN-120574+-expressing T cells CD25+Foxp3+ regulatoryT cells and CD20+IL-10+-producing B cells were assessedby estimating positively staining cells and results werereported as the percentage of immunoreactive cells Resultsare expressed as the mean plusmn SEM of cells quantified by theprogram ImagePro Plus version 511 [16]

26 Flow Cytometry A sample of venous blood (10mL) wasobtained from each subject Peripheral blood mononuclearcells (PBMCs) were isolated by gradient centrifugation onLymphoprep (Axis-Shield PoC AS Oslo Norway)

ForTh22(CD3+CD4+CD161minusIL-22+)Th17(CD3+CD4+CD161+IL-17A+) Th2 (CD3+ CD4+ CD14minusIL-4+) Th1(CD3+CD4+CD14minusIFN-120574+) and regulatory T cells (CD3+CD4+CD25+Foxp3+) PBMCs were labelled with 5 120583L ofanti-human CD3-FITC- anti-CD4-PeCy5- and anti-CD161-APC-conjugated monoclonal antibodies (BD BiosciencesSan Jose CA) anti-CD14-FITC- anti-CD4-PeCy5- anti-CD3-APC- or CD3-FITC- anti-CD4-PeCy5- and anti-CD25-APC-conjugated monoclonal antibodies (BD Bio-sciences San Jose CA) Intracellular staining was per-formed with 5 120583L of an anti-human IL-22-PE- IL-17A-PE-IL-4-PE- IFN-120574-PE- or anti-human Foxp3-PE-labelled

monoclonal antibodies (BD Biosciences) Finally T sub-sets were analyzed by flow cytometry with an AccuriC6 (BD Biosciences) A total of 500000ndash1000000 eventswere recorded for each sample and analyzed with theFlowJo X software (Tree Star Inc) An electronic gatewas made for CD3+CD4+CD161minus CD3+CD4+CD161+CD3+CD14minusCD4+ cells or CD3+CD4+CD25hi Resultsare expressed as the relative percentage of IL-22- IL-17A- IL-4- IFN-120574- or Foxp3-expressing cells in each gate

As isotype control IgG1-FITCIgG

1-PECD45-PeCy5

mouse IgG1120581 (BD Tritest BD Biosciences) was used to set

the threshold and gates in the cytometerWe ran an unstained(autofluorescence control) and permeabilized PBMCs sam-ple Autofluorescence control was compared to single stainedcell positive controls to confirm that the stained cells wereon scale for each parameter Besides BD Calibrite 3 beadswere used to adjust instrument settings set fluorescence com-pensation and check instrument sensitivity (BD CaliBRITEBD Biosciences) Fluorescence minus one (FMO) controlswere stained in parallel using the panel of antibodies withsequential omission of one antibody [20]

27 Circulating Neurologic Autoantibodies Serum antibodieswere evaluated with a commercially available kit Neurol-ogy Mosaic 1 (Euroimmun Lubeck Germany) by stan-dard indirect immunofluorescence screening assay using asantigenic substrate frozen monkey nerves cerebellum andintestinal tissue following manufacturerrsquos instructions Serafrom patients and controls diluted at 110 were incubatedon intestinal cerebellum and peripheral nerves of monkeytissue sections A positive anti-Hu serumwas used as controlFluorescein-labelled anti-human immunoglobulin G (IgG)conjugate was used as secondary antibody Slides were exam-ined on a fluorescence photomicroscope (times200 and times400)Anti-Hu-positive serum labelled neuron nuclei on bothcerebellum and myenteric plexus sections For circulatingneurologic antibodies positivity staining of both nucleus andcytoplasm of myenteric plexus neurons was considered [21]

28 Immunoblot Analysis To further analyze target antigensof circulating anti-myenteric autoantibodies sera were testedwith the neuronal antigens profile plus RST (Euroimmun)This test is a membrane strip with a combination of neu-ronal antigens (amphiphysin CV2 and PNMA2 (Ma-2Ta))onconeural antigens (Ri Yo and Hu) recoverin SOX-1and titin separately After blot strip blocking sera wereincubated at 1100 for 1 hour at room temperature To detectthe bound antibodies a second incubation was carried outusing alkaline phosphatase-labelled antihuman IgG For theinterpretation a EUROLine Scan software (Euroimmun) wasused [21]

29 In Situ Hybridization for HSV-1 Paraffin-embeddedspecimens of lower esophageal sphincter muscle from acha-lasia patients and controls were deparaffinized for 18 h at60∘C and sequentially immersed in xylene (30min at 37∘C)absolute ethanol 75 ethanol 50 ethanol 25 ethanol andwater Cells were permeabilized and incubated with 1mgLproteinase K (Promega Madison WI USA) for 30min at


37∘C Codenaturation and hybridization were done by sepa-ration of double-strand DNA and binding of single-strandedprobe HSV-1 FITC-conjugated PNA probe mixture (DakoDenmark) was applied to the specimen on each slide and theywere sealed and incubated for 3min at 73∘C then for 4 hrs at37∘C and finally for 2 hrs at 55∘C Probe detection was doneby the addition of an anti-FITCAP antibody and alkalinephosphatase Positive cells were considered if the purplereaction product was seen Tissues were counterstained withnuclear fast red The negative controls included 5 biopsies ofesophagectomy from patients with squamous cell carcinomaand were identified as noninfected patients (negative forHSV-1 DNA)

210 RT-PCR for HSV Detection of HSV-1 DNA was per-formed by reverse transcriptase and PCR in specimens fromlower esophageal sphincter muscle from achalasia patientsand controls Four 120583m thick sections of available formalin-fixed paraffin embedded tissue were deparaffinized and rehy-drated Cells were permeabilized and incubated with 1mgLproteinase K (Promega Madison WI USA) for 30min at37∘C Tissue was treated with DNase I (Invitrogen AlamedaCA USA) and slides were sealed with the assembly tooland placed in a thermocycler (Perkin Elmer CambridgeUK) RT was done by incubation of the sections with 50 ngrandom primers 1120583L 10mM dNTPs water 15 120583L 4 120583L 5xfirst-strand buffer 2 120583L 01M DTT 1 120583L RNase OUT and200U of M-MLV RT (Invitrogen Alameda CA USA) ina thermocycler (Perkin Elmer) at 37∘C for 50min PCRreaction was performed with 1x reaction buffer (Invitro-gen Alameda CA USA) 15 U Taq polymerase 2mmolLMgCl

2 40mmolL dNTP 0sdot2mmolL dUTP-digoxigenin

(Boehringer Mannheim Lewes UK) and 60 pg each ofHSV-1 primers (51015840 CATCACCGACCCGGAGAGGGAC31015840(sense) and 51015840GGGCCAGGCGCTTGTTGGTGTA31015840 (anti-sense))The slides were sealed with the assembly tool (PerkinElmer) and placed in a Touchdown Thermocycler (PerkinElmer Cambridge UK) cDNA was amplified as followsdenaturation at 95∘C for 1min annealing at 70∘C for 1minand extension at 72∘C for 1min for 35 sequential cyclesPCR products were detected with alkaline phosphatase-conjugated sheep antibodies against digoxigenin (RocheMannheim Germany) diluted 1500 and the chromogenwas 5-bromo-4-chloro-3-3 indolyl phosphate toluidine saltand tetrazolium nitroblue (Boehringer Manheim) diluted150 Sections were counterstained with nuclear fast red ormalachite green to avoid any interference with the blue signalgenerated by HSV DNA To avoid bias one section from oneblock from each individual was used in masked assays andit was scored positive if the blue reaction product was seenOne section from a patient with infection HSV-1 was used aspositive control The negative controls included 5 biopsies ofesophagectomy from patients with squamous cell carcinomaand were identified as noninfected patients (negative forHSV-1 DNA)

211 Statistical Analysis Descriptive statistics were per-formed and categorical variables were compared using 1198832

test or Fisherrsquos exact test for analysis of continuous variablesStudent 119905-test was employed One-way analysis of variance onranks by Holm-Sidak method was performed for both pair-wise comparisons and comparison versus a control groupStatistical analysis was done using the SigmaStat112 program(Aspire Software International Leesburg VA USA) Datawere expressed as the median range and mean plusmn standarddeviation (SD)standard error of the mean (SEM) The 119875values smaller than or equal to 005 were considered assignificant This study conforms to STROBE statement alongwith references to STROBE and the broader EQUATORguidelines [22]

212 Study Approval This work was performed accordingto the principles expressed in the Declaration of HelsinkiThe study was approved by the ethical committee from ourinstitution and awritten informed consentwas obtained fromall subjects

3 Results

31 Demographic Data The study included 32 patients withidiopathic achalasia Twenty-five percent of patients weretype I 59 type II and 16 type III achalasia The meanage of the patients was similar among the 3 types Althoughthe female to male ratio was similar in type I (44) therewas a difference in sex ratio in types II (127) and III (41)Two patients from type II achalasia group also had SjogrenrsquosSyndrome and Guillain-Barre Syndrome respectively Twopatients from type III achalasia group also had SjogrenrsquosSyndrome and ankylosing spondylitis Demographic andclinical variables and laboratory data from the healthy donors(PBMCs) tissue control group (esophageal biopsies) and theachalasia patients are described in Table 1

32 Histological Findings Esophageal muscle biopsies fromprimary achalasia patients had abundant perineural inflam-matory infiltrates as well as an increase of endoneurialconnective tissue and myopathic changes Histopathologicalanalysis showed capillaritis (51) plexitis (23) venulitis(7) fibrosis (3) and nerve hypertrophy (16) (Figures1(a) and 1(b))

33 Proteins Involved in Extracellular Matrix TurnoverMMP-9 also known as 92 kDa gelatinase is involved in extra-cellular matrix degradation in physiological and pathologicalprocesses The myenteric plexus of esophageal tissue fromachalasia patients showed a statistically significant increase inMMP-9 percentage versus tissue control group Nonethelessthe number of MMP-9+ cells in types II and III achalasia washigher when compared to type I achalasia (Table 2 Figures1(c) and 1(d))

TIMP-1 inhibits not only the proteolytic activity butalso apoptosis and induces cell proliferation TIMP-1+ cellpercentage was higher in achalasia patients compared withcontrol group Moreover number of TIMP-1-producing cellsin types II and III achalasia was higher when compared totype I achalasia (Table 2 Figures 1(e) and 1(f))


Table 1 Demographic clinical and laboratory variables

Blood healthy donors Tissue control group Type I achalasia Type II achalasia Type III achalasia(n = 20) (n = 5) (n = 8) (n = 19) (n = 5)

Age (years) mean plusmn SD 408 plusmn 101 722 plusmn 89lowast 442 plusmn 142 456 plusmn 163 450 plusmn 174Sex femalemale 128 41 44 127 41Disease evolution (months) mean plusmn SD ND ND 437 plusmn 122 369 plusmn 79 690 plusmn 358Dysphagia () ND ND 100 95 100Regurgitation () ND ND 86 94 80Weight loss () ND ND 71 78 100Pyrosis () ND ND 57 42 40Other autoimmune diseases () ND ND 0 11 40

lowast

ESR gt20mmHg () ND ND 17 25 20lowast

119875 lt 005 ND Not determined

34 Expression of Pro-InflammatoryAnti-Fibrogenic Cytok-ines IL-22 belongs to the IL-10 superfamily It initiates innateimmune response against pathogens in gut epithelial andrespiratory cells and regulates antibody production IL-22+cell percentage was conspicuously increased in myentericplexus of esophageal tissue samples when compared withcontrol group However IL-22+ cell number was increased intype II and type III achalasia versus type I achalasia (Table 2Figures 2(a) and 2(b))

IL-17 is defined as key mediator of inflammatory autoim-mune diseases It promotes recruitment of neutrophils acti-vation of innate immune cells enhances B-cell functions andinduces proinflammatory cytokines (TNF-120572 IL-1120573) IL-17A+cell frequency was higher in myenteric plexus of esophagealachalasia tissue patients versus control tissue Moreover ahigher immunoreactive cell numberwas found in types II andIII achalasia when compared with type I achalasia (Table 2Figures 2(c) and 2(d))

Achalasia patients had significantly higher percentage ofIFN-120574CD4 T cells versus control group Nonetheless levelsof IFN-120574CD4 T cells were higher in types II and III achalasiacompared with type I achalasia (Table 2 Figures 2(e) and2(f))

35 Expression of Anti-InflammatoryProfibrogenic CytokinesTGF-1205731 accomplishes many cellular functions including con-trol of cellular growth proliferation differentiation negativeregulation of inflammation collagen synthesis and apop-tosis Thus myenteric plexus of esophageal biopsies fromidiopathic achalasia patients showed a significant increase onTGF-1205731+ cell expression compared with control tissue It isimportant to highlight that types I and III achalasia patientshad higher percentage of immunoreactive cells versus type II(Table 2 Figures 3(a) and 3(b))

IL-4 is an anti-inflammatory cytokine that inhibits thesynthesis of IL-1120573 TNF-120572 IL-6 IL-17A and so forth Itregulates B-cell proliferation and differentiation and it is alsoa potent inhibitor of apoptosis IL-4 is synthesized primarilybyTh2 cells Achalasia patients had significantly higher IL-4+cell percentage when comparedwith control group HoweverIL-4+ cell number was higher in type II and type III achalasiaversus type I achalasia (Table 2 Figures 3(c) and 3(d))

IL-13 has similar functions to IL-4 However it alsoregulates type I collagen gene and thus participates in fibrosisType II achalasia patients showed an increase in IL-13-producing cells when compared with control tissue and typesI and III achalasia patients (Table 2 Figures 3(e) and 3(f))

36 Regulatory Cells Regulatory T cells (Tregs) are a spe-cialized subpopulation of T cells that suppress the activationof effector T cells They maintain homeostasis and promotetolerance to self-antigens Achalasia patients showed a higherTreg frequency in myenteric plexus of esophageal tissuecompared with tissue controls Nonetheless the highest Tregcell percentage was found in type III achalasia versus types Iand II (Table 2 Figures 4(a) and 4(b))

Recently the presence of a B cell subpopulation withregulatory capacity (Bregs) has been shown in humans Thissuppressive function has been attributed to IL-10 produc-tion Furthermore Bregs promote T-cell differentiation to aregulatory phenotype and induce remission of autoimmunemanifestations in murine models The myenteric plexus ofesophageal biopsies obtained from achalasia patients had ahigher relative IL-10-producing B-cell percentage when com-pared with tissues from control group It is noteworthy thattype III achalasia patients showed a statistically significantincrease levels versus types I and II achalasia (Table 2 Figures4(c) and 4(d))

37 Fas Expression FAS receptor also known as apopto-sis antigen 1 (CD95) is a 36KDa surface protein witha cytoplasmic domain of ldquocell deathrdquo Its binding to FASligand induces complete apoptosis The myenteric plexusof esophageal biopsies from achalasia patients had higherFas-expressing cell frequency versus control tissue Type IIIachalasia patients had highest Fas+ cell percentage whencompared with types II and I achalasia (Table 2 Figures 4(e)and 4(f))

38 Percentage of Circulating CD4+ T-Cell SubpopulationsTo determine the different T-cell subpopulations PBMCswere immunophenotyped and analyzed by flow cytometryRelative percentage of circulating Th22 and Th17 cells fromachalasia patients was higher when compared with healthyindividuals Type III achalasia patients showed an increment


Tissue architectureControl Achalasia

(a)

Plexitis23

Nervehypertrophy

16

Venulitis7

Fibrosis3

Capillaritis51

(b)

MMP-9Control Achalasia

(c)

MM

P-9

imm

unor

eact

ive c

ells

()

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0005 0002

MeanMedianControl (n = 5)

Type I achalasia (n = 8)

Type II achalasia (n = 13)

Type III achalasia (n = 5)

(d)

TIMP-1Control Achalasia

times320

(e)

TIM

P-1

imm

unor

eact

ive c

ells

()

0

10

20

30

40

50

600015 0035

0010 lt0001 lt0001





(f)

Figure 1 Proteins involved in extracellularmatrix turnover in achalasia (a) Representative photomicrograph froma control and an idiopathicachalasia tissue in which an abundant inflammatory infiltrate is observed Original magnification was times200 (b) Histological findings inbiopsies from achalasia patients (c) Immunoreactive cells Original magnification was times320 (d) Results are expressed as mean (yellow line)median (black line) and 5th95th percentiles of MMP-9+ cells (e) Immunohistochemistry for TIMP-1 in control and achalasia sectionsArrows depict immunoreactive cells Original magnification was times320 (f) Results are expressed as mean (black line) median (yellow line)and 5th95th percentiles of TIMP-1+ cells


Table 2 Percentage of immunoreactive cells in achalasia patients

Control group Type I achalasia Type II achalasia Type III achalasia(n = 5) (n = 8) (n = 13) (n = 5)

Extracellular matrix turnover proteinsMMP-9 ()

Mean plusmn SEM 136 plusmn 24 318 plusmn 22lowastlowastlowast 385 plusmn 09lowastlowastlowast 410 plusmn 15lowastlowastlowast

Median 145 290 385 405Range 2ndash25 23ndash45 29ndash47 35ndash48

TIMP-1 ()Mean plusmn SEM 141 plusmn 15 259 plusmn 24lowastlowastlowast 348 plusmn 16lowastlowastlowast 351 plusmn 44lowastlowastlowast


Proinflammatory cytokinesIL-22 ()



IL-17A ()Mean plusmn SEM 47 plusmn 08 195 plusmn 15lowastlowastlowast 228 plusmn 06lowastlowastlowast 273 plusmn 13lowastlowastlowast


CD4IFN-120574 ()Mean plusmn SEM 38 plusmn 06 235 plusmn 11lowastlowastlowast 280 plusmn 07lowastlowastlowast 268 plusmn 07lowastlowastlowast


Profibrogenic cytokinesTGF-1205731 ()



IL-4 ()Mean plusmn SEM 109 plusmn 16 212 plusmn 22lowastlowastlowast 328 plusmn 10lowastlowastlowast 314 plusmn 09lowastlowastlowast




Regulatory cellsCD4Foxp3 ()



CD20IL-10 ()Mean plusmn SEM 80 plusmn 12 237 plusmn 11lowastlowastlowast 226 plusmn 08lowastlowastlowast 287 plusmn 29lowastlowastlowast


ApoptosisFAS ()


Median 10 270 300 350


Table 2 Continued


Range 3ndash19 23ndash36 11ndash41 22ndash56lowast

119875 lt 005 control versus achalasia patientslowastlowast

119875 lt 001 control versus achalasia patientslowastlowastlowast

119875 lt 0001 control versus achalasia patients

versus type I and II achalasia patients (Table 3 Figures 5(a)and 5(b))

Regarding theTh2 cells there was a significant increase intype III achalasia patients versus type I (Table 3 Figure 5(c))

On the other hand relative percentage of Th1 cells washigher in type II achalasia patients when compared with typeI (Table 3 Figure 5(d))

Finally Tregs showed a statistically significant increaseonly in type III achalasia patients (Table 3 Figure 5(e))

39 Antibodies against Myenteric Plexus The prevalence ofnuclear or cytoplasmic circulating autoantibodies againstmyenteric plexus in the sera from idiopathic achalasiapatientswas 100 (1414) comparedwith healthy donors (0)(Figure 6(a)) Most antibodies were positive against nucleiand nucleolus in myenteric plexus (Figure 6(a))

310 Immunoblot Analysis By immunoblot analysis it wasdetermined that 69 (913) of sera were positive to PNMA2(Ma-2Ta) antigen Only one serum was labelled to recoverin(Figure 6(b))

311 In Situ Viral Infection and Active Replication of HSV-1 HSV-1 DNA (viral infection) RNA (active replication)and virus were detected in all tissues (1414) from achalasiapatients by PCR (Figure 7(a)) RT-PCR (Figure 7(b)) andimmunohistochemistry (Figure 7(c)) but not in control tis-sues It is noteworthy that only one (114) and two (214)achalasia patients were cytomegalovirus- and Epstein-Barrvirus-positive respectively (data not shown)

4 Discussion

Idiopathic achalasia is an inflammatory disease caused by theloss of the inhibitory neurons of the esophageal myentericplexus Etiology of the myenteric plexus inflammation is stillunknown [23] Nevertheless based on the evidence obtainedin our research it is possible to postulate an etio- andphysiopathogenic mechanism for achalasia

Initial event that triggers disease appears to be theresult of a repetitive insult produced by neurotropic virusinfection likely HSV-1 [12 13 24ndash26] which induces aconspicuous and persistent inflammation at the perineu-ral level in Auerbachrsquos plexus Not all infected patientsdevelop achalasia Thus it is not preposterous to suggestthat only those individuals with genetic predisposition todevelop a chronic autoinflammatory response will progressto the disease [13] In fact there are many studies that

showed a positive association for the class II HLA antigenincluding DQw1 DQA1lowast0103 DQB1lowast0601 DQB1lowast0602DQB1lowast0603 DQB1lowast0601 DQB1lowast0502 and DQB1lowast0503alleles in Caucasians [4 17 27ndash29] Furthermore it has beendemonstrated in 1068 cases of achalasia from central EuropeSpain and Italy that an eight-residue insertion at position227ndash234 in the cytoplasmic tail of HLA-DQ1205731 (encodedby HLA-DQB1lowast0503 and HLA-DQB1lowast0601) confers thestrongest risk for achalasia In addition two amino acidsubstitutions in the extracellular domain of HLA-DQ1205721at position 41 (lysine encoded by HLA-DQA1lowast0103) andof HLA-DQ1205731 at position 45 (glutamic acid encoded byHLA-DQB1lowast0301 and HLA-DQB1lowast0304) independentlyconfer achalasia risk [29]

In addition destruction of inhibitory ganglionic cells dueto the abundant autoimmune inflammatory infiltrates hyper-trophy and neuronal fibrosis during disease progression isfound [30] In most of the cases pathology is accompaniedby the presence of neuronal autoantibodies [18 21] thatcontribute to the destruction of the myenteric plexus [31 32]as previously has been demonstrated by Bruley des Varanneset al where serum from achalasia patients but not fromgastroesophageal reflux disease can induce phenotypic andfunctional changes in myenteric neurons which reproducethe characteristics of the disease and discard the fact thatthese autoantibodies could be an epiphenomenon [2]

In this study we determined in situ viral infection andactive replication of HSV-1 but not CMV or EBV (data notshown) along myenteric plexus HSV-1 preferential ports ofentry are represented by the perioral and the esophagealmucosa Furthermore herpes viruses exhibit a strong tropismfor nerve fibers and following the primary exposure theviruses remain in a latent form in the nucleus of neurons[12] Thus one of the potential triggers for the developmentof immune-mediated diseases is an infection In particularin autoimmune diseases characterized by immune-mediatedinflammation to self-antigens it has been established thatviral or bacterial infections may trigger the cascade of eventsin genetic susceptible patients Viral infection induce anincrease in the amount of T-cell population infiltrates whencomparedwith the controls and healthy donors locally (biop-sies) and systemically (circulating cells) To our knowledgethis is the first study that evaluates the presence of twoCD4T-cell subpopulations involved in autoimmune inflammationTh22 and Th17 cells Th17 cells synthesize IL-17A IL-17FIL-21 and so forth and have been demonstrated in autoim-mune disease such as Sjogrenrsquos syndrome multiple sclerosispsoriasis autoimmune uveitis juvenile diabetes rheumatoid


IL-22Control Achalasia

(a)

IL-2

2 im

mun

orea

ctiv

e cel

ls (

)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

lt0001 lt0001





(b)


(c)

IL-1

7A im

mun

orea

ctiv

e cel

ls (

)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0014 0003

lt0001





(d)

Control AchalasiaCD4IFN-120574

times320

(e)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

lt0001 0049

CD4

IFN

-120574im

mun

orea

ctiv

e cel

ls (

)





(f)

Figure 2 Proinflammatoryantifibrogenic cytokine expression in achalasia Immunohistochemistry for (a) IL-22 (c) IL-17 and (e) CD4IFN-120574 T cells in control and achalasia sections Arrows depict immunoreactive cells Original magnification was times320 Results are expressed asmean (yellow line) median (black line) and 5th95th percentiles of (b) IL-22+ (d) IL-17+ and (f) CD4IFN-120574+ T cells


Control AchalasiaTGF-1205731

(a)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0008 0023

TGF-1205731

imm

unor

eact

ive c

ells

()





(b)

IL- 4Control Achalasia

(c)

0

10

20

30

40

50

60

lt0001

lt0001 lt0001 lt0001

lt0001

IL-4

imm

unor

eact

ive c

ells

()





(d)


times320

(e)

0

10

20

30

40

50

60

lt0001 0015

lt0001 lt0001 lt0001

IL-1

3 im

mun

orea

ctiv

e cel

ls (

)





(f)

Figure 3 Anti-inflammatoryprofibrogenic cytokine expression in achalasia Immunohistochemistry for (a) TGF-1205731 (c) IL-4 and (e) IL-13cells in control and achalasia sections Arrows depict immunoreactive cells Original magnification was times320 Results are expressed as mean(yellow line) median (black line) and 5th95th percentiles of (b) TGF-1205731+ (d) IL-4+ and (f) IL-13+ cells


CD25Foxp3Control Achalasia

(a)

0

10

20

30

40

50

60

CD25

Fox

p3 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0001

0001





(b)

CD20IL-10Control Achalasia

(c)

0

10

20

30

40

50

60

CD20

IL-1

0 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0006

0049





(d)

FASControl Achalasia

times320

(e)

0

10

20

30

40

50

60

FAS

imm

unor

eact

ive c

ells

()

lt0001 lt0001 lt0001

0008

0006





(f)

Figure 4 Regulatory cells and apoptosis in achalasia Immunohistochemistry for (a) CD25Foxp3 T cells (c) CD20IL-10 B cells and (e)FAS cells in control and achalasia sections Arrows depict immunoreactive cells Original magnification was times320 Results are expressed asmean (yellow line) median (black line) and 5th95th percentiles of (b) CD25Foxp3+ T cells (d) CD20IL-10+ B cells and (f) FAS+ cells


Table 3 Percentage of Th22- Th17- Th2- Th1- and Tregs-circulating cells in achalasia patients

Healthy donors Type I achalasia Type II achalasia Type III achalasia(n = 20) (n = 6) (n = 8) (n = 5)

IL-22-expressing T cells(Th22 )CD3+CD4+CD161minusIL-22+


Median 14 61 57 113

Range 04ndash27 51ndash89 33ndash94 95ndash138

IL-17A-expressing T cells(Th17 )CD3+CD4+CD161+IL-17A+


Median 17 41 64 100



Mean plusmn SEM 16 plusmn 02 23 plusmn 11 50 plusmn 05lowastlowastlowast 36 plusmn 05lowastlowastlowast

Median 16 25 47 42


IFN-120574-expressing T cells(Th1 )CD3+CD4+CD14minusIFN-120574+


Median 17 26 32 37


Foxp3-expressing T cells(Tregs )CD3+CD4+CD25hiFoxp3+

Mean plusmn SEM 52 plusmn 04 43 plusmn 06 59 plusmn 06 107 plusmn 09lowastlowastlowast

Median 46 42 61 108





arthritis and Crohnrsquos disease [15 16 20 33 34] On the otherhandTh22 synthesizes primarily IL-22 It plays an importantrole in the pathogenesis of many autoimmune disorderssuch as psoriasis rheumatoid arthritis Sjogrenrsquos syndromehepatitis graft versus host disease and allergic diseases [1635] IL-17A-and IL-22-producing cells were the main cellularcomponents of the inflammatory foci in achalasia tissue Inaddition an increase in relative percentage of circulatingTh17 andTh22 cells was observed in achalasia patients whencompared with healthy controls These data strongly suggestthat achalasia is also an autoimmune disease Moreover fourof our achalasia patients had other autoimmune diseases such

as Sjogrenrsquos syndrome (2) ankylosing spondylitis (1) andGuillain-Barre syndrome (1) suggesting that achalasia has anautoimmune component [4] Finally it has been reported thata severe recurrent achalasia cardia patient who responded totreatment of autoimmune acquired hemophilia with high-dose steroid therapy administered for 7 months withoutrecurrence of the achalasia for more than 2 years Thusresponse of the achalasia to immunosuppressive therapy alsosuggests that achalasia is an autoimmune disorder [36]

Regarding types of achalasia we hypothesized (a) thattype III could be a more aggressive disease (b) that type IIachalasia is an earlier stage of a continuum natural history


Th22

()

0

2

4

6

8

10

12

14

16

lt0001 lt0001 lt0001

lt0001

lt0001

(a)Th

17 (

)

lt0001 lt0001 lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

0038

(b)

Th2

()

lt0001 0002

lt0001

0

2

4

6

8

10

12

14

16

0047

0035

(c)

Th1

()

lt0001 lt0001

0032

0

2

4

6

8

10

12

14

16

(d)

Treg

s (

)lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

MeanMedian



Type III achalasia (n = 5)Healthy donors (n = 20)

(e)

Figure 5 CirculatingCD4+ T-cell subsets in achalasia Relative percentage of circulating (a)Th22 (b)Th17 (c)Th2 (d)Th1 and (e) regulatoryT cells Results are expressed as mean (yellow line) median (black line) and 5th95th percentiles of T cells

of the disease that evolves into type I whereas type IIIcould represent the earliest stage of the disease or (c) thattype III could be a disease with different physiopathology[5 37] In this context we determine that achalasia type IIhas an increment of IL-22 IL-17ATh1 cell percentage and IL-4- and IL-13-producing cells (potentially anti-inflammatoryand profibrogenic cytokines resp) when compared withtype I achalasia This supports that type II achalasia is anactive pathologic process in an earlier stage of the diseaseWhereas in situ IL-17A- CD25Foxp3- CD20IL-10- andFAS-producing cells and Th22 Th17 and Tregs peripheralcells are significantly higher in type III achalasia whencompared with the other groups Moreover the concomitantpresence of inflammatory (IL-17 IL-22) and regulatory cells(Tregs and IL-10-producing B cells) suggests that the proin-flammatory and regulatory balance favors the former withthe incapacity of the latter in trying to maintain homeostasisConversely the as yet unknown persistent inflammatorytrigger surpasses the regulatory potential of Foxp3 T cells

and CD20IL-10 B cells conducting to a more vigorous tissuedamage characterized by an increase of MMP-9 and TIMP-1

Finally significant increase of autoantibodies againstmyenteric plexus in all the patients with achalasia alsosupports the hypothesis of an autoimmune mechanism Inorder to characterize protein targets of circulating anti-myenteric autoantibodies we used immunoblot analysis onprimate cerebellum and myenteric plexus protein extract toestablish whether achalasia sera with nuclear reactivity bindto proteins of a givenmolecular weight A 69 of sera reactedwith PNMA2Ta (Ma2Ta) and 8 reacted with recoverinantigen (Figure 6) To our knowledge this is the first studythat describes the specificity of anti-myenteric autoantibodiesto Ma2Ta protein which are related to Sjogrenrsquos syndromeHowever we do not discard the possibility of the presence ofother autoantibodies with different antigenic specificity forexample GAD 65 [18]

There are potential limitations to the current study Firststudies of HLA association in Mexican Mestizo patients are


Negative control Positive control Patient (n = 13)

200x

(a)

Ma2Ta

Positive control

(b)

Figure 6 Anti-myenteric plexus antibodies and antigenicity (a) Indirect immunofluorescence of monkey intestinal tissue containingAuerbachrsquos plexus Arrows show positive fluorescent nuclei and cytoplasm of cells that were recognized by circulating anti-myenteric plexusautoantibodies from achalasia patients (right panel) (b) Profile of neuronal antigens (antigens on membrane strips) Arrow in upper stripshows circulating autoantibodies against PNMA2 (Ma2Ta) antigen Arrow in lower strip shows circulating antibodies against recoverinantigen

needed for a better understanding of the disease Second thiswork was a cross-sectional study Thus longitudinal studiesare required to provide the basis for the search of specificbiomarkers that contribute to early diagnosis of the disease[38]

Summing up our results suggest that achalasia is adisease with an important local inflammatory and systemicautoimmune component [35] This autoimmune responseappears to be associated with the presence of HSV-1 andrelated to the high prevalence of specific autoantibodiesagainst the myenteric plexus (Figure 8)

Our results shed further light into the preponderantautoimmune inflammation and the possible role of viralinfection and certainly deserve to be studied in depth in orderto evaluate the nature of genetic predisposition to the diseasedevelopment Our data may contribute to the identificationof important disease cell and cytokine targets in achalasiawhich finally may result in improved therapeutic manage-ment for example immunosuppressive therapy Moreoverour findings may provide the basis for the search of specificbiomarkers that contribute to early diagnosis of the disease

Significance of This Study

What Is Already Known onThis Subject

(i) Idiopathic achalasia is a rare esophageal motor disor-der characterized by loss of myenteric plexus Acha-lasia is postulated to be an autoimmune disease

(ii) The presence of circulating anti-myenteric autoanti-bodies has been reported

(iii) Some studies have implicated viral agents in theetiology and pathogenesis of the disease

What Are the New Findings

(i) An increased percentage of two CD4+ T-cell sub-populations related to autoimmune diseases Th17and Th22 were identified in peripheral cells andtheir cytokines were detected in the inflammatoryinfiltrates of the lower esophageal sphincter biopsiesfrom achalasia patients

(ii) Circulating anti-myenteric autoantibodies predom-inantly directed against PNMA2 (Ma-2Ta) antigenwere detected in sera from achalasia patients

(iii) HSV-1 DNA (viral infection) RNA (active replica-tion) and virus were detected in the lower esophagealsphincter biopsies from achalasia patients

How Might It Impact on Clinical Practice inthe Foreseeable Future

(i) Our data may contribute to the identification ofimportant disease cell and cytokine targets in achala-sia which finally may result in improved therapeuticmanagement for example immunosuppressive ther-apy


In situ hybridization for HSV-1

Negative control HSV-1

times200

(a)

RT-PCR for HSV-1

Positive control Control tissue HSV-1

times200

(b)

Immunohistochemistry

times320

(c)

Figure 7 HSV-1 identification (a) In situ hybridization Arrows depict achalasia tissue with viral infection (b) In situ RT-PCR for activereplication of HSV-1 Arrows show antigen detection of HSV-1 infected cells in a skin lesion (positive control) and achalasia tissueOriginal magnification was times200 (c) Immunohistochemistry for antigen HSV-1 detection Arrows depict immunoreactive cells Originalmagnification was times320


pDCTh1Genetic predisposition

Nonautoimmuneinflammatory infiltrates

Th1 TregsBregs

pDCregs

Humoral response(anti-Ma2Ta anti-recoverin

autoantibodies etc

Apoptosis ofneurons

Chronic infectious insult(HSV-1 varicella zoster

measles bornavirus etc)

Loss of immunological tolerance

Autoimmune inflammatory infiltrates

Th22Th17TregsBregs

pDCregs

Myenteric neuron abnormalities

PlexitisGanglionitisVasculitis

Absence of peristalsisImpaired relaxation of

lower esophageal sphincter

Extracellular turnoverWound repair

FibrosisMMPTIMP-1

Th1Th2

TregsBregspDCs

Th22

Th1ThThBreg

pDCTh2Th2

pDC

gggggggggggggg

Th2

Th2Th1

pDCTh2

Th1

Th17Th22

Th17

Breg

B

Th1ThThBreg

pDC Th1Th1Th1Th11

Th1Th2

Achalasia 8

7

6

5

4

3

1

2

4

TGF-1205731IL-4IL-13

(HLA-DQ1205731 DQ1205721 etc)

Treg

Treg

Treg

Treg

Treg

Treg

2998400

2998400 Breg

Th17Th22

Figure 8 Proposed model of achalasia pathophysiology (1) Active or latent infectious insult in achalasia is strongly suggested by severalstudies Some neurotropic viruses such as the herpes family of viruses have predilection for squamous epithelium andmay cause ganglion celldamage that is limited to the esophagus (2) Some individuals with genetic predisposition will develop an aggressive inflammatory response(3) At very early stage of the disease it is possible that inflammatory infiltrates may be predominantly composed of Th1 Th2 and regulatorycell subsets (Tregs Bregs and plasmacytoid dendritic cells (pDCs)) (4) Repair of tissue after injury requires orchestrated coordination ofseveral cell types and biosynthetic processes and is coordinated by an interacting group of pro- and anti-inflammatory cytokines fibrousextracellular matrix (ECM) proteins to replace lost or damaged tissue and products of metabolism such as oxygen radicals The mostprominent profibrogenic cytokines are TGF120573 IL-4 and IL-13 ECM alsomediates cellular crosstalk and does so in two ways Newly depositedECM is then rebuilding over time to emulate normal tissue Matrix proteinases and their inhibitors (TIMPs) also are important both duringwound repair tissue remodeling and fibrosis (21015840 5) If steps 1ndash4 happen repeatedly chronic infection only those individuals with geneticpredisposition to develop a long-lasting autoinflammatory response will progress to develop the disease (loss of peripheral tolerance) Thusautoinflammatory infiltrates are predominantly composed ofTh22Th17 and regulatory subpopulations (6) Degeneration and significant lossof nerve fibers associated with autoinflammatory infiltrates of the myenteric plexus provide evidence of an immune mediated destructionof the inhibitory neurons not only by necrosis but also by apoptosis (FasFasL overexpression) (7) Autoimmune etiology of achalasia isfurther supported by the presence of anti-myenteric autoantibodies in sera (8) Pathophysiologically achalasia is cause by autoinflammationdegeneration of nerves in the esophagus plexitis abnormalities inmicrovasculature ganglionitis and finally by the loss of inhibitory ganglionin the myenteric plexus

(ii) Our findings may provide the basis for the search ofspecific biomarkers that contribute to early diagnosisof the disease

Abbreviations

AP Alkaline phosphataseAPC AllophycocyaninBregs Regulatory B cellsCD Cluster differentiationcDNA Complementary DNA

DAB DiaminobenzidinedNTPs Deoxyribonucleotide triphosphateDTT DithiothreitoldUTP Deoxyuridine triphosphateESR Erythrocyte sedimentation rateFITC Fluorescein isothiocyanateFMO Fluorescence minus oneFoxp3 Forkhead box P3HCV Hepatitis C virusHD Healthy donorsHIV Human immunodeficiency virus


HRM High-resolution manometryHRP Horseradish peroxidaseHSV-1 Herpes simplex virus-1IFN-120574 Interferon-gammaIL InterleukinLES Lower esophageal sphincterLINK Dextran polymer coupled with secondary

antibodies against mouse and rabbitimmunoglobulins

MgCl2 Magnesium chloride

M-MLV Moloney murine Leukemia virus enzymeMMP-9 Matrix metalloproteinase-9PBMCs Peripheral blood mononuclear cellsPCR Polymerase chain reactionPE PhycoerythrinPeCy5 Phycoerythrin cychrome 5RT Reverse transcriptionSD Standard deviationSEM Standard error of the meanTGF-1205731 Transforming growth factor-betaTh T helper cellsTIMP-1 Tissue inhibitor of metalloproteinase-1Tregs Regulatory T cells

Conflict of Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contribution

J Furuzawa-Carballeda is responsible for study concept anddesign acquisition of data analysis and interpretation of datadrafting of the paper critical revision of the paper for impor-tant intellectual content statistical analysis and technicalsupport and obtained funding D Aguilar-Leon contributedto the acquisition of data analysis and interpretation ofdata critical revision of the paper for important intellec-tual content and technical support A Gamboa-Domınguezperformed study design acquisition of data analysis andinterpretation of data critical revision of the paper forimportant intellectual content and technical support M AValdovinos contributed to study design analysis and inter-pretation of data critical revision of the paper for importantintellectual content and technical support C Nunez-Alvarezcontributed to acquisition of data analysis and interpretationof data critical revision of the paper for important intellectualcontent and technical support L A Martın-del-Campo andE Coss-Adame contributed to acquisition of data analysisand interpretation of data critical revision of the paper forimportant intellectual content and technical and materialsupport A B Enrıquez A E Svarch A Flores-Najera AVilla-Banos and J C Ceballos contributed to acquisitionof data analysis and interpretation of data and technicaland material support G Torres-Villalobos is responsiblefor study concept and design acquisition of data analysisand interpretation of data drafting of the paper criticalrevision of the paper for important intellectual contentstatistical analysis technical support and study supervisionand obtained funding

References

[1] G E Boeckxstaens ldquoNovel mechanism for impaired nitrergicrelaxation in achalasiardquo Gut vol 55 no 3 pp 304ndash305 2006

[2] S Bruley des Varannes J Chevalier S Pimont et al ldquoSerumfrom achalasia patients alters neurochemical coding in themyenteric plexus and nitric oxide mediated motor response innormal human fundusrdquo Gut vol 55 no 3 pp 319ndash326 2006

[3] J F Mayberry ldquoEpidemiology and demographics of achalasiardquoGastrointestinal Endoscopy Clinics of North America vol 11 no2 pp 235ndash247 2001

[4] G E Boeckxstaens G Zaninotto and J E Richter ldquoAchalasiardquoThe Lancet vol 383 no 9911 pp 83ndash93 2014

[5] J E Pandolfino M A Kwiatek T Nealis W Bulsiewicz JPost and P J Kahrilas ldquoAchalasia a new clinically relevantclassification by high-resolutionmanometryrdquoGastroenterologyvol 135 no 5 pp 1526ndash1533 2008

[6] T Willis Pharmaceutice Rationalis Sive Diatribe de Medica-mentorum Operationibus in Humano Corpore Hagae ComitisLondon UK 1674

[7] I Gockel J R E Bohl S Doostkam V F Eckardt and TJunginger ldquoSpectrum of histopathologic findings in patientswith achalasia reflects different etiologiesrdquo Journal of Gastroen-terology and Hepatology vol 21 no 4 pp 727ndash733 2006

[8] U C Ghoshal S B Daschakraborty and R Singh ldquoPathogen-esis of achalasia cardiardquoWorld Journal of Gastroenterology vol18 no 24 pp 3050ndash3057 2012

[9] R P Petersen A V Martin C A Pellegrini and B KOelschlager ldquoSynopsis of investigations into proposed theorieson the etiology of achalasiardquo Diseases of the Esophagus vol 25no 4 pp 305ndash310 2012

[10] A Kilic AM Krasinskas S R Owens J D Luketich R J Lan-dreneau and M J Schuchert ldquoVariations in inflammation andnerve fiber loss reflect different subsets of achalasia patientsrdquoJournal of Surgical Research vol 143 no 1 pp 177ndash182 2007

[11] L Raymond B Lach and F M Shamji ldquoInflammatory aetiol-ogy of primary oesophageal achalasia an immunohistochemi-cal and ultrastructural study of Auerbachrsquos plexusrdquoHistopathol-ogy vol 35 no 5 pp 445ndash453 1999

[12] M Facco P Brun I Baesso et al ldquoT cells in the myentericplexus of achalasia patients show a skewed TCR repertoire andreact to HSV-1 antigensrdquo American Journal of Gastroenterologyvol 103 no 7 pp 1598ndash1609 2008

[13] G E Boeckxstaens ldquoAchalasia virus-induced euthanasia ofneuronsrdquo The American Journal of Gastroenterology vol 103no 7 pp 1610ndash1612 2008

[14] S B Clark T W Rice R R Tubbs J E Richter and J RGoldblum ldquoThe nature of the myenteric infiltrate in achalasiaan immunohistochemical analysisrdquo The American Journal ofSurgical Pathology vol 24 no 8 pp 1153ndash1158 2000

[15] G Fonseca-Camarillo E Mendivil-Rangel J Furuzawa-Carballeda and J K Yamamoto-Furusho ldquoInterleukin 17gene and protein expression are increased in patients withulcerative colitisrdquo Inflammatory Bowel Diseases vol 17 no 10pp E135ndashE136 2011

[16] J Furuzawa-Carballeda J Sanchez-Guerrero J L Betanzoset al ldquoDifferential cytokine expression and regulatory cellsin patients with primary and secondary Sjogrenrsquos syndromerdquoScandinavian Journal of Immunology vol 80 no 6 pp 432ndash4402014


[17] A Ruiz-de-Leon J Mendoza C Sevilla-Mantilla et al ldquoMyen-teric antiplexus antibodies and class II HLA in achalasiardquoDigestive Diseases and Sciences vol 47 no 1 pp 15ndash19 2002

[18] R E Kraichely G Farrugia S J Pittock D O Castell and VA Lennon ldquoNeural autoantibody profile of primary achalasiardquoDigestive Diseases and Sciences vol 55 no 2 pp 307ndash311 2010

[19] A J Bredenoord M Fox P J Kahrilas J E Pandolfino WSchwizer and A J P M Smout ldquoChicago classification criteriaof esophageal motility disorders defined in high resolutionesophageal pressure topographyrdquo Neurogastroenterology andMotility vol 24 supplement 1 pp 57ndash65 2012

[20] J Furuzawa-Carballeda G Fonseca-Camarillo G Lima andJ K Yamamoto-Furusho ldquoIndoleamine 23-dioxygenaseexpressing cells in inflammatory bowel diseasemdasha cross-sectional studyrdquo Clinical and Developmental Immunology vol2013 Article ID 278035 14 pages 2013

[21] M Kallel-Sellami S Karoui H Romdhane et al ldquoCirculatingantimyenteric autoantibodies in Tunisian patients with idio-pathic achalasiardquo Diseases of the Esophagus vol 26 no 8 pp782ndash787 2013

[22] I Simera D Moher J Hoey K F Schulz and D G Altman ldquoAcatalogue of reporting guidelines for health researchrdquo EuropeanJournal of Clinical Investigation vol 40 no 1 pp 35ndash53 2010

[23] W Park and M F Vaezi ldquoEtiology and pathogenesis ofachalasia the current understandingrdquoThe American Journal ofGastroenterology vol 100 no 6 pp 1404ndash1414 2005

[24] H Niwamoto E Okamoto J Fujimoto M Takeuchi J-IFuruyama and Y Yamamoto ldquoAre human herpes viruses ormeasles virus associated with esophageal achalasiardquo DigestiveDiseases and Sciences vol 40 no 4 pp 859ndash864 1995

[25] E Sinagra E Gallo F Mocciaro et al ldquoJC virus helicobacterpylori and oesophageal achalasia preliminary results from aretrospective case-control studyrdquo Digestive Diseases and Sci-ences vol 58 no 5 pp 1433ndash1434 2013

[26] D Ganem A Kistler and J DeRisi ldquoAchalasia and viralinfection new insights from veterinary medicinerdquo ScienceTranslational Medicine vol 2 no 33 Article ID 33ps24 2010

[27] A Latiano R de Giorgio U Volta et al ldquoHLA and entericantineuronal antibodies in patients with achalasiardquo Neurogas-troenterology amp Motility vol 18 no 7 pp 520ndash525 2006

[28] R KHWong C LMaydonovitch S JMetz and J R Baker JrldquoSignificant DQw1 association in achalasiardquo Digestive Diseasesand Sciences vol 34 no 3 pp 349ndash352 1989

[29] I Gockel J Becker M M Wouters et al ldquoCommon variantsin the HLA-DQ region confer susceptibility to idiopathicachalasiardquo Nature Genetics vol 46 no 8 pp 901ndash904 2014

[30] H Akiho E Ihara YMotomura and K Nakamura ldquoCytokine-induced alterations of gastrointestinal motility in gastrointesti-nal disordersrdquo World Journal of Gastrointestinal Pathophysiol-ogy vol 2 no 5 pp 72ndash81 2011

[31] R Dhamija K M Tan S J Pittock A Foxx-Orenstein EBenarroch and V A Lennon ldquoSerologic profiles aiding thediagnosis of autoimmune gastrointestinal dysmotilityrdquo ClinicalGastroenterology and Hepatology vol 6 no 9 pp 988ndash9922008

[32] P L Moses L M Ellis M R Anees et al ldquoAntineuronal anti-bodies in idiopathic achalasia and gastro-oesophageal refluxdiseaserdquo Gut vol 52 no 5 pp 629ndash636 2003

[33] T Korn E Bettelli M Oukka and V K Kuchroo ldquoIL-17 andTh17 cellsrdquo Annual Review of Immunology vol 27 pp 485ndash5172009

[34] J Furuzawa-Carballeda G Lima J Jakez-Ocampo and LLlorente ldquoIndoleamine 23-dioxygenase-expressing peripheralcells in rheumatoid arthritis and systemic lupus erythematosusa cross-sectional studyrdquo European Journal of Clinical Investiga-tion vol 41 no 10 pp 1037ndash1046 2011

[35] X Yang and S G Zheng ldquoInterleukin-22 a likely target fortreatment of autoimmune diseasesrdquoAutoimmunity Reviews vol13 no 6 pp 615ndash620 2014

[36] H Al-Jafar M Laffan S Al-Sabah M Elmorsi M Habeeband F Alnajar ldquoSevere recurrent achalasia cardia responding totreatment of severe autoimmune acquired haemophiliardquo CaseReports in Gastroenterology vol 6 no 3 pp 618ndash623 2012

[37] P J Kahrilas and G Boeckxstaens ldquoThe spectrum of achalasialessons from studies of pathophysiology and high-resolutionmanometryrdquoGastroenterology vol 145 no 5 pp 954ndash965 2013

[38] I Gockel M Muller and J Schumacher ldquoAchalasiamdasha diseaseof unknown cause that is often diagnosed too laterdquo DeutschesArzteblatt International vol 109 no 12 pp 209ndash214 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



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OphthalmologyJournal of


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Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


(HRM) as the gold standard while the esophagram withbarium swallow and endoscopy is useful to rule out otherpathologies Idiopathic achalasia has been classified in threetypes based on the HRM achalasia with minimal esophagealpressurization (type I) achalasia with esophageal compres-sion (type II) and achalasia with spasm (type III) [5]

Even though the disease was first described morethan 300 years ago etiology and physiopathology are stillunknown However many contemporary hypotheses suggesta multifactorial etiopathogenic mechanism [6 7] Thuspathophysiologically idiopathic achalasia is caused by loss ofinhibitory ganglion in the myenteric plexus of the esophagus[7ndash11] In order to explain this several studies have attemptedto explore initiating agents that may cause the disease suchas latent or active viral infection Proposal virus candidateshave included herpes simplex virus (HSV) (neurotropic viruswith a predilection for squamous epithelium) bornavirusvaricella zoster (VZ) measles and human papilloma virus(HPV) [12 13] Nonetheless not all the patients with viralinfections develop the disease Numerous potential genetictargets exist in the immune system which may createsusceptibility Some studies have been shown genetic cor-relations in achalasia for example ALADIN gen associ-ated with Allgrove syndrome receptor of vasoactive intesti-nal polypeptide (VIPR1) gene polymorphism interleukin-(IL-) 23 receptor polymorphism protein tyrosine phos-phatase nonreceptor 22 gene polymorphism and humanleukocyte antigen- (HLA-) DQ1205731 (119867119871119860-1198631198761198611lowast0503 and119867119871119860-1198631198761198611lowast0601) HLA-DQ1205721 (119867119871119860-1198631198761198601lowast0103) andHLA-DQ1205731 (119867119871119860-1198631198761198611lowast0301 and119867119871119860-1198631198761198611lowast0304) [8]Consequently chronic viral infection could trigger aberrantimmune response under appropriate genetic and environ-mental background allowing the loss of esophageal neuronsMoreover detailed examination of Auerbachrsquos plexus hasshown infiltration of CD3+CD45RO+ T cells predominantlyCD8+ T cytotoxic lymphocytes expressing activationmarkers[7 10 11 14] Notwithstanding little is known about whetherthese subpopulations of cells are truly autoimmune as inthe case of pathogenic Th22 and Th17 effector cell subsetsand what happens with cellular mechanisms which areparticipating in an attempt to regulate the inflammationsuch as regulatory CD4+ T cells and IL-10-producing B cells[15 16] Finally in accordance with the hypotheses evidenceautoantibodies against myenteric neurons have been shownin serum samples from patients with achalasia especiallyin those with HLA DQA1lowast0103 and DQB1lowast0603 alleles[17] However no specific antigenicity of myenteric neuronautoantibodies has been identified [2 18]

Based on these data and hypotheses the aim of the studywas to analyze in esophageal muscle tissue and peripheralblood the localized and systemic inflammation patterns inidiopathic achalasia patients Furthermore we evaluate theexistence of circulating anti-myenteric autoantibodies andtheir specificities in sera frompatients and assess the presenceof herpes simplex virus type 1 (HSV-1) in esophageal muscle

2 Materials and Methods

21 Study Design This was an exploratory observationaland cross-sectional study designed to identify by the first

time the presence of CD4+ T-cell subpopulations relatedto autoimmune diseases autoantibodies and their speci-ficity and proteins involved in extracellular matrix turnoverapoptosis proinflammatory and profibrogenic cytokinesand regulatory T and B cells (Tregs and Bregs) in loweresophageal sphincter biopsies from achalasia patientsThirty-two patients with idiopathic achalasia without Chagas dis-ease HIV or HCV infections were included between January2013 and July 2014 The medical records of each patientwere reviewed Peripheral blood was obtained from 20 age-matched healthy donors (HD) who volunteered at the bloodbank HDs were also interviewed in order to discard any dis-ease including known autoimmune disease use of immuno-suppressors or prednisone and concurrent infections Fivebiopsies of esophagectomy from patients with squamous cellcarcinoma from upper third of esophagus were included ascontrol tissue samples (see Supplementary Figure 1 in Supple-mentaryMaterial available online at httpdxdoiorg1011552015729217)

22HighResolution EsophagealManometry Protocol Ahigh-resolution esophageal manometry was performed in everypatient at baseline and before being referred to surgeryA solid-state HRM probe with 36 circumferential sensorswas used (Given Imaging Yokneam Israel) Having thepatient in a sitting position and at 45 degrees stationaryHMR was performed After a 12 hr fasting period the probewas inserted transnasally until passing the esophagogastricjunction assessed visually in the computer screen Ten waterswallows of 5mL separated by 30 seconds were provided

Analyses were performed using Manoview 20 (GivenImaging) and patients were classified according to latestChicago classification [19] into three groups (1) type I acha-lasia (without pressurization within the esophageal body)(2) type II (with pan-pressurization) and (3) type III (withspasm) Classification was performed by two gastroenterol-ogists (MAV E-CA) experts in high-resolution esophagealmanometry Surgeon was blinded to the high-resolutionmanometry analysis [19]

23 Erythrocyte Sedimentation Rate Erythrocyte sedimenta-tion rate (ESR) was determined by Westergren method

24 Immunohistochemistry Once the myotomy was done a2 cm long and 2mm width tissue was obtained Tissue wasimmediately formalin-fixed and paraffin embedded Four120583m thick tissue sections were deparaffinized and rehydratedTissues were incubated for 18 h at 4∘C with goat polyclonalanti-human MMP-9 TIMP-1 TGF-1205731 IL-22 IL-17A FASor mouse monoclonal IgG

1HSV-1 antibody (Santa Cruz

Biotechnology Santa Cruz CA USA) at 10120583gmL Bind-ing was detected by incubating sections with biotinylateddonkey anti-goat IgG antibody (ABC Staining System SantaCruz Biotechnology) Slides were incubated with horseradishperoxidase- (HRP-) streptavidin and 331015840-diaminobenzidine(DAB) (Sigma-Aldrich) and counterstained with hema-toxylin Negative control staining was performed with nor-mal human serumdiluted 1 100 instead of primary antibodyThe reactive blank was incubated with phosphate buffer










1-PECD45-PeCy5














3 Results









lowast















(a)

Plexitis23

Nervehypertrophy

16

Venulitis7

Fibrosis3

Capillaritis51

(b)


(c)

MM

P-9

imm

unor

eact

ive c

ells

()

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0005 0002





(d)


times320

(e)

TIM

P-1

imm

unor

eact

ive c

ells

()

0

10

20

30

40

50

600015 0035

0010 lt0001 lt0001





(f)





























ApoptosisFAS ()


Median 10 270 300 350


Table 2 Continued













4 Discussion








(a)

IL-2

2 im

mun

orea

ctiv

e cel

ls (

)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

lt0001 lt0001





(b)


(c)

IL-1

7A im

mun

orea

ctiv

e cel

ls (

)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0014 0003

lt0001





(d)


times320

(e)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

lt0001 0049

CD4

IFN

-120574im

mun

orea

ctiv

e cel

ls (

)





(f)




(a)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0008 0023

TGF-1205731

imm

unor

eact

ive c

ells

()





(b)


(c)

0

10

20

30

40

50

60

lt0001

lt0001 lt0001 lt0001

lt0001

IL-4

imm

unor

eact

ive c

ells

()





(d)


times320

(e)

0

10

20

30

40

50

60

lt0001 0015

lt0001 lt0001 lt0001

IL-1

3 im

mun

orea

ctiv

e cel

ls (

)





(f)




(a)

0

10

20

30

40

50

60

CD25

Fox

p3 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0001

0001





(b)


(c)

0

10

20

30

40

50

60

CD20

IL-1

0 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0006

0049





(d)


times320

(e)

0

10

20

30

40

50

60

FAS

imm

unor

eact

ive c

ells

()

lt0001 lt0001 lt0001

0008

0006





(f)







Median 14 61 57 113




Median 17 41 64 100




Median 16 25 47 42




Median 17 26 32 37




Median 46 42 61 108









Th22

()

0

2

4

6

8

10

12

14

16

lt0001 lt0001 lt0001

lt0001

lt0001

(a)Th

17 (

)

lt0001 lt0001 lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

0038

(b)

Th2

()

lt0001 0002

lt0001

0

2

4

6

8

10

12

14

16

0047

0035

(c)

Th1

()

lt0001 lt0001

0032

0

2

4

6

8

10

12

14

16

(d)

Treg

s (

)lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

MeanMedian




(e)








200x

(a)

Ma2Ta

Positive control

(b)



















times200

(a)

RT-PCR for HSV-1


times200

(b)


times320

(c)





Th1 TregsBregs

pDCregs


autoantibodies etc

Apoptosis ofneurons





Th22Th17TregsBregs

pDCregs






FibrosisMMPTIMP-1

Th1Th2

TregsBregspDCs

Th22

Th1ThThBreg

pDCTh2Th2

pDC

gggggggggggggg

Th2

Th2Th1

pDCTh2

Th1

Th17Th22

Th17

Breg

B

Th1ThThBreg

pDC Th1Th1Th1Th11

Th1Th2

Achalasia 8

7

6

5

4

3

1

2

4

TGF-1205731IL-4IL-13

(HLA-DQ1205731 DQ1205721 etc)

Treg

Treg

Treg

Treg

Treg

Treg

2998400

2998400 Breg

Th17Th22



Abbreviations












References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























1-PECD45-PeCy5














3 Results









lowast















(a)

Plexitis23

Nervehypertrophy

16

Venulitis7

Fibrosis3

Capillaritis51

(b)


(c)

MM

P-9

imm

unor

eact

ive c

ells

()

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0005 0002





(d)


times320

(e)

TIM

P-1

imm

unor

eact

ive c

ells

()

0

10

20

30

40

50

600015 0035

0010 lt0001 lt0001





(f)





























ApoptosisFAS ()


Median 10 270 300 350


Table 2 Continued













4 Discussion








(a)

IL-2

2 im

mun

orea

ctiv

e cel

ls (

)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

lt0001 lt0001





(b)


(c)

IL-1

7A im

mun

orea

ctiv

e cel

ls (

)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0014 0003

lt0001





(d)


times320

(e)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

lt0001 0049

CD4

IFN

-120574im

mun

orea

ctiv

e cel

ls (

)





(f)




(a)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0008 0023

TGF-1205731

imm

unor

eact

ive c

ells

()





(b)


(c)

0

10

20

30

40

50

60

lt0001

lt0001 lt0001 lt0001

lt0001

IL-4

imm

unor

eact

ive c

ells

()





(d)


times320

(e)

0

10

20

30

40

50

60

lt0001 0015

lt0001 lt0001 lt0001

IL-1

3 im

mun

orea

ctiv

e cel

ls (

)





(f)




(a)

0

10

20

30

40

50

60

CD25

Fox

p3 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0001

0001





(b)


(c)

0

10

20

30

40

50

60

CD20

IL-1

0 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0006

0049





(d)


times320

(e)

0

10

20

30

40

50

60

FAS

imm

unor

eact

ive c

ells

()

lt0001 lt0001 lt0001

0008

0006





(f)







Median 14 61 57 113




Median 17 41 64 100




Median 16 25 47 42




Median 17 26 32 37




Median 46 42 61 108









Th22

()

0

2

4

6

8

10

12

14

16

lt0001 lt0001 lt0001

lt0001

lt0001

(a)Th

17 (

)

lt0001 lt0001 lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

0038

(b)

Th2

()

lt0001 0002

lt0001

0

2

4

6

8

10

12

14

16

0047

0035

(c)

Th1

()

lt0001 lt0001

0032

0

2

4

6

8

10

12

14

16

(d)

Treg

s (

)lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

MeanMedian




(e)








200x

(a)

Ma2Ta

Positive control

(b)



















times200

(a)

RT-PCR for HSV-1


times200

(b)


times320

(c)





Th1 TregsBregs

pDCregs


autoantibodies etc

Apoptosis ofneurons





Th22Th17TregsBregs

pDCregs






FibrosisMMPTIMP-1

Th1Th2

TregsBregspDCs

Th22

Th1ThThBreg

pDCTh2Th2

pDC

gggggggggggggg

Th2

Th2Th1

pDCTh2

Th1

Th17Th22

Th17

Breg

B

Th1ThThBreg

pDC Th1Th1Th1Th11

Th1Th2

Achalasia 8

7

6

5

4

3

1

2

4

TGF-1205731IL-4IL-13

(HLA-DQ1205731 DQ1205721 etc)

Treg

Treg

Treg

Treg

Treg

Treg

2998400

2998400 Breg

Th17Th22



Abbreviations












References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























3 Results









lowast















(a)

Plexitis23

Nervehypertrophy

16

Venulitis7

Fibrosis3

Capillaritis51

(b)


(c)

MM

P-9

imm

unor

eact

ive c

ells

()

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0005 0002





(d)


times320

(e)

TIM

P-1

imm

unor

eact

ive c

ells

()

0

10

20

30

40

50

600015 0035

0010 lt0001 lt0001





(f)





























ApoptosisFAS ()


Median 10 270 300 350


Table 2 Continued













4 Discussion








(a)

IL-2

2 im

mun

orea

ctiv

e cel

ls (

)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

lt0001 lt0001





(b)


(c)

IL-1

7A im

mun

orea

ctiv

e cel

ls (

)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0014 0003

lt0001





(d)


times320

(e)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

lt0001 0049

CD4

IFN

-120574im

mun

orea

ctiv

e cel

ls (

)





(f)




(a)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0008 0023

TGF-1205731

imm

unor

eact

ive c

ells

()





(b)


(c)

0

10

20

30

40

50

60

lt0001

lt0001 lt0001 lt0001

lt0001

IL-4

imm

unor

eact

ive c

ells

()





(d)


times320

(e)

0

10

20

30

40

50

60

lt0001 0015

lt0001 lt0001 lt0001

IL-1

3 im

mun

orea

ctiv

e cel

ls (

)





(f)




(a)

0

10

20

30

40

50

60

CD25

Fox

p3 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0001

0001





(b)


(c)

0

10

20

30

40

50

60

CD20

IL-1

0 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0006

0049





(d)


times320

(e)

0

10

20

30

40

50

60

FAS

imm

unor

eact

ive c

ells

()

lt0001 lt0001 lt0001

0008

0006





(f)







Median 14 61 57 113




Median 17 41 64 100




Median 16 25 47 42




Median 17 26 32 37




Median 46 42 61 108









Th22

()

0

2

4

6

8

10

12

14

16

lt0001 lt0001 lt0001

lt0001

lt0001

(a)Th

17 (

)

lt0001 lt0001 lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

0038

(b)

Th2

()

lt0001 0002

lt0001

0

2

4

6

8

10

12

14

16

0047

0035

(c)

Th1

()

lt0001 lt0001

0032

0

2

4

6

8

10

12

14

16

(d)

Treg

s (

)lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

MeanMedian




(e)








200x

(a)

Ma2Ta

Positive control

(b)



















times200

(a)

RT-PCR for HSV-1


times200

(b)


times320

(c)





Th1 TregsBregs

pDCregs


autoantibodies etc

Apoptosis ofneurons





Th22Th17TregsBregs

pDCregs






FibrosisMMPTIMP-1

Th1Th2

TregsBregspDCs

Th22

Th1ThThBreg

pDCTh2Th2

pDC

gggggggggggggg

Th2

Th2Th1

pDCTh2

Th1

Th17Th22

Th17

Breg

B

Th1ThThBreg

pDC Th1Th1Th1Th11

Th1Th2

Achalasia 8

7

6

5

4

3

1

2

4

TGF-1205731IL-4IL-13

(HLA-DQ1205731 DQ1205721 etc)

Treg

Treg

Treg

Treg

Treg

Treg

2998400

2998400 Breg

Th17Th22



Abbreviations












References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















lowast















(a)

Plexitis23

Nervehypertrophy

16

Venulitis7

Fibrosis3

Capillaritis51

(b)


(c)

MM

P-9

imm

unor

eact

ive c

ells

()

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0005 0002





(d)


times320

(e)

TIM

P-1

imm

unor

eact

ive c

ells

()

0

10

20

30

40

50

600015 0035

0010 lt0001 lt0001





(f)





























ApoptosisFAS ()


Median 10 270 300 350


Table 2 Continued













4 Discussion








(a)

IL-2

2 im

mun

orea

ctiv

e cel

ls (

)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

lt0001 lt0001





(b)


(c)

IL-1

7A im

mun

orea

ctiv

e cel

ls (

)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0014 0003

lt0001





(d)


times320

(e)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

lt0001 0049

CD4

IFN

-120574im

mun

orea

ctiv

e cel

ls (

)





(f)




(a)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0008 0023

TGF-1205731

imm

unor

eact

ive c

ells

()





(b)


(c)

0

10

20

30

40

50

60

lt0001

lt0001 lt0001 lt0001

lt0001

IL-4

imm

unor

eact

ive c

ells

()





(d)


times320

(e)

0

10

20

30

40

50

60

lt0001 0015

lt0001 lt0001 lt0001

IL-1

3 im

mun

orea

ctiv

e cel

ls (

)





(f)




(a)

0

10

20

30

40

50

60

CD25

Fox

p3 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0001

0001





(b)


(c)

0

10

20

30

40

50

60

CD20

IL-1

0 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0006

0049





(d)


times320

(e)

0

10

20

30

40

50

60

FAS

imm

unor

eact

ive c

ells

()

lt0001 lt0001 lt0001

0008

0006





(f)







Median 14 61 57 113




Median 17 41 64 100




Median 16 25 47 42




Median 17 26 32 37




Median 46 42 61 108









Th22

()

0

2

4

6

8

10

12

14

16

lt0001 lt0001 lt0001

lt0001

lt0001

(a)Th

17 (

)

lt0001 lt0001 lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

0038

(b)

Th2

()

lt0001 0002

lt0001

0

2

4

6

8

10

12

14

16

0047

0035

(c)

Th1

()

lt0001 lt0001

0032

0

2

4

6

8

10

12

14

16

(d)

Treg

s (

)lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

MeanMedian




(e)








200x

(a)

Ma2Ta

Positive control

(b)



















times200

(a)

RT-PCR for HSV-1


times200

(b)


times320

(c)





Th1 TregsBregs

pDCregs


autoantibodies etc

Apoptosis ofneurons





Th22Th17TregsBregs

pDCregs






FibrosisMMPTIMP-1

Th1Th2

TregsBregspDCs

Th22

Th1ThThBreg

pDCTh2Th2

pDC

gggggggggggggg

Th2

Th2Th1

pDCTh2

Th1

Th17Th22

Th17

Breg

B

Th1ThThBreg

pDC Th1Th1Th1Th11

Th1Th2

Achalasia 8

7

6

5

4

3

1

2

4

TGF-1205731IL-4IL-13

(HLA-DQ1205731 DQ1205721 etc)

Treg

Treg

Treg

Treg

Treg

Treg

2998400

2998400 Breg

Th17Th22



Abbreviations












References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)

Plexitis23

Nervehypertrophy

16

Venulitis7

Fibrosis3

Capillaritis51

(b)


(c)

MM

P-9

imm

unor

eact

ive c

ells

()

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0005 0002





(d)


times320

(e)

TIM

P-1

imm

unor

eact

ive c

ells

()

0

10

20

30

40

50

600015 0035

0010 lt0001 lt0001





(f)





























ApoptosisFAS ()


Median 10 270 300 350


Table 2 Continued













4 Discussion








(a)

IL-2

2 im

mun

orea

ctiv

e cel

ls (

)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

lt0001 lt0001





(b)


(c)

IL-1

7A im

mun

orea

ctiv

e cel

ls (

)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0014 0003

lt0001





(d)


times320

(e)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

lt0001 0049

CD4

IFN

-120574im

mun

orea

ctiv

e cel

ls (

)





(f)




(a)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0008 0023

TGF-1205731

imm

unor

eact

ive c

ells

()





(b)


(c)

0

10

20

30

40

50

60

lt0001

lt0001 lt0001 lt0001

lt0001

IL-4

imm

unor

eact

ive c

ells

()





(d)


times320

(e)

0

10

20

30

40

50

60

lt0001 0015

lt0001 lt0001 lt0001

IL-1

3 im

mun

orea

ctiv

e cel

ls (

)





(f)




(a)

0

10

20

30

40

50

60

CD25

Fox

p3 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0001

0001





(b)


(c)

0

10

20

30

40

50

60

CD20

IL-1

0 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0006

0049





(d)


times320

(e)

0

10

20

30

40

50

60

FAS

imm

unor

eact

ive c

ells

()

lt0001 lt0001 lt0001

0008

0006





(f)







Median 14 61 57 113




Median 17 41 64 100




Median 16 25 47 42




Median 17 26 32 37




Median 46 42 61 108









Th22

()

0

2

4

6

8

10

12

14

16

lt0001 lt0001 lt0001

lt0001

lt0001

(a)Th

17 (

)

lt0001 lt0001 lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

0038

(b)

Th2

()

lt0001 0002

lt0001

0

2

4

6

8

10

12

14

16

0047

0035

(c)

Th1

()

lt0001 lt0001

0032

0

2

4

6

8

10

12

14

16

(d)

Treg

s (

)lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

MeanMedian




(e)








200x

(a)

Ma2Ta

Positive control

(b)



















times200

(a)

RT-PCR for HSV-1


times200

(b)


times320

(c)





Th1 TregsBregs

pDCregs


autoantibodies etc

Apoptosis ofneurons





Th22Th17TregsBregs

pDCregs






FibrosisMMPTIMP-1

Th1Th2

TregsBregspDCs

Th22

Th1ThThBreg

pDCTh2Th2

pDC

gggggggggggggg

Th2

Th2Th1

pDCTh2

Th1

Th17Th22

Th17

Breg

B

Th1ThThBreg

pDC Th1Th1Th1Th11

Th1Th2

Achalasia 8

7

6

5

4

3

1

2

4

TGF-1205731IL-4IL-13

(HLA-DQ1205731 DQ1205721 etc)

Treg

Treg

Treg

Treg

Treg

Treg

2998400

2998400 Breg

Th17Th22



Abbreviations












References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































ApoptosisFAS ()


Median 10 270 300 350


Table 2 Continued













4 Discussion








(a)

IL-2

2 im

mun

orea

ctiv

e cel

ls (

)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

lt0001 lt0001





(b)


(c)

IL-1

7A im

mun

orea

ctiv

e cel

ls (

)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0014 0003

lt0001





(d)


times320

(e)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

lt0001 0049

CD4

IFN

-120574im

mun

orea

ctiv

e cel

ls (

)





(f)




(a)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0008 0023

TGF-1205731

imm

unor

eact

ive c

ells

()





(b)


(c)

0

10

20

30

40

50

60

lt0001

lt0001 lt0001 lt0001

lt0001

IL-4

imm

unor

eact

ive c

ells

()





(d)


times320

(e)

0

10

20

30

40

50

60

lt0001 0015

lt0001 lt0001 lt0001

IL-1

3 im

mun

orea

ctiv

e cel

ls (

)





(f)




(a)

0

10

20

30

40

50

60

CD25

Fox

p3 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0001

0001





(b)


(c)

0

10

20

30

40

50

60

CD20

IL-1

0 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0006

0049





(d)


times320

(e)

0

10

20

30

40

50

60

FAS

imm

unor

eact

ive c

ells

()

lt0001 lt0001 lt0001

0008

0006





(f)







Median 14 61 57 113




Median 17 41 64 100




Median 16 25 47 42




Median 17 26 32 37




Median 46 42 61 108









Th22

()

0

2

4

6

8

10

12

14

16

lt0001 lt0001 lt0001

lt0001

lt0001

(a)Th

17 (

)

lt0001 lt0001 lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

0038

(b)

Th2

()

lt0001 0002

lt0001

0

2

4

6

8

10

12

14

16

0047

0035

(c)

Th1

()

lt0001 lt0001

0032

0

2

4

6

8

10

12

14

16

(d)

Treg

s (

)lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

MeanMedian




(e)








200x

(a)

Ma2Ta

Positive control

(b)



















times200

(a)

RT-PCR for HSV-1


times200

(b)


times320

(c)





Th1 TregsBregs

pDCregs


autoantibodies etc

Apoptosis ofneurons





Th22Th17TregsBregs

pDCregs






FibrosisMMPTIMP-1

Th1Th2

TregsBregspDCs

Th22

Th1ThThBreg

pDCTh2Th2

pDC

gggggggggggggg

Th2

Th2Th1

pDCTh2

Th1

Th17Th22

Th17

Breg

B

Th1ThThBreg

pDC Th1Th1Th1Th11

Th1Th2

Achalasia 8

7

6

5

4

3

1

2

4

TGF-1205731IL-4IL-13

(HLA-DQ1205731 DQ1205721 etc)

Treg

Treg

Treg

Treg

Treg

Treg

2998400

2998400 Breg

Th17Th22



Abbreviations












References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 2 Continued













4 Discussion








(a)

IL-2

2 im

mun

orea

ctiv

e cel

ls (

)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

lt0001 lt0001





(b)


(c)

IL-1

7A im

mun

orea

ctiv

e cel

ls (

)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0014 0003

lt0001





(d)


times320

(e)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

lt0001 0049

CD4

IFN

-120574im

mun

orea

ctiv

e cel

ls (

)





(f)




(a)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0008 0023

TGF-1205731

imm

unor

eact

ive c

ells

()





(b)


(c)

0

10

20

30

40

50

60

lt0001

lt0001 lt0001 lt0001

lt0001

IL-4

imm

unor

eact

ive c

ells

()





(d)


times320

(e)

0

10

20

30

40

50

60

lt0001 0015

lt0001 lt0001 lt0001

IL-1

3 im

mun

orea

ctiv

e cel

ls (

)





(f)




(a)

0

10

20

30

40

50

60

CD25

Fox

p3 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0001

0001





(b)


(c)

0

10

20

30

40

50

60

CD20

IL-1

0 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0006

0049





(d)


times320

(e)

0

10

20

30

40

50

60

FAS

imm

unor

eact

ive c

ells

()

lt0001 lt0001 lt0001

0008

0006





(f)







Median 14 61 57 113




Median 17 41 64 100




Median 16 25 47 42




Median 17 26 32 37




Median 46 42 61 108









Th22

()

0

2

4

6

8

10

12

14

16

lt0001 lt0001 lt0001

lt0001

lt0001

(a)Th

17 (

)

lt0001 lt0001 lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

0038

(b)

Th2

()

lt0001 0002

lt0001

0

2

4

6

8

10

12

14

16

0047

0035

(c)

Th1

()

lt0001 lt0001

0032

0

2

4

6

8

10

12

14

16

(d)

Treg

s (

)lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

MeanMedian




(e)








200x

(a)

Ma2Ta

Positive control

(b)



















times200

(a)

RT-PCR for HSV-1


times200

(b)


times320

(c)





Th1 TregsBregs

pDCregs


autoantibodies etc

Apoptosis ofneurons





Th22Th17TregsBregs

pDCregs






FibrosisMMPTIMP-1

Th1Th2

TregsBregspDCs

Th22

Th1ThThBreg

pDCTh2Th2

pDC

gggggggggggggg

Th2

Th2Th1

pDCTh2

Th1

Th17Th22

Th17

Breg

B

Th1ThThBreg

pDC Th1Th1Th1Th11

Th1Th2

Achalasia 8

7

6

5

4

3

1

2

4

TGF-1205731IL-4IL-13

(HLA-DQ1205731 DQ1205721 etc)

Treg

Treg

Treg

Treg

Treg

Treg

2998400

2998400 Breg

Th17Th22



Abbreviations












References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)

IL-2

2 im

mun

orea

ctiv

e cel

ls (

)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

lt0001 lt0001





(b)


(c)

IL-1

7A im

mun

orea

ctiv

e cel

ls (

)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0014 0003

lt0001





(d)


times320

(e)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

lt0001 0049

CD4

IFN

-120574im

mun

orea

ctiv

e cel

ls (

)





(f)




(a)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0008 0023

TGF-1205731

imm

unor

eact

ive c

ells

()





(b)


(c)

0

10

20

30

40

50

60

lt0001

lt0001 lt0001 lt0001

lt0001

IL-4

imm

unor

eact

ive c

ells

()





(d)


times320

(e)

0

10

20

30

40

50

60

lt0001 0015

lt0001 lt0001 lt0001

IL-1

3 im

mun

orea

ctiv

e cel

ls (

)





(f)




(a)

0

10

20

30

40

50

60

CD25

Fox

p3 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0001

0001





(b)


(c)

0

10

20

30

40

50

60

CD20

IL-1

0 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0006

0049





(d)


times320

(e)

0

10

20

30

40

50

60

FAS

imm

unor

eact

ive c

ells

()

lt0001 lt0001 lt0001

0008

0006





(f)







Median 14 61 57 113




Median 17 41 64 100




Median 16 25 47 42




Median 17 26 32 37




Median 46 42 61 108









Th22

()

0

2

4

6

8

10

12

14

16

lt0001 lt0001 lt0001

lt0001

lt0001

(a)Th

17 (

)

lt0001 lt0001 lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

0038

(b)

Th2

()

lt0001 0002

lt0001

0

2

4

6

8

10

12

14

16

0047

0035

(c)

Th1

()

lt0001 lt0001

0032

0

2

4

6

8

10

12

14

16

(d)

Treg

s (

)lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

MeanMedian




(e)








200x

(a)

Ma2Ta

Positive control

(b)



















times200

(a)

RT-PCR for HSV-1


times200

(b)


times320

(c)





Th1 TregsBregs

pDCregs


autoantibodies etc

Apoptosis ofneurons





Th22Th17TregsBregs

pDCregs






FibrosisMMPTIMP-1

Th1Th2

TregsBregspDCs

Th22

Th1ThThBreg

pDCTh2Th2

pDC

gggggggggggggg

Th2

Th2Th1

pDCTh2

Th1

Th17Th22

Th17

Breg

B

Th1ThThBreg

pDC Th1Th1Th1Th11

Th1Th2

Achalasia 8

7

6

5

4

3

1

2

4

TGF-1205731IL-4IL-13

(HLA-DQ1205731 DQ1205721 etc)

Treg

Treg

Treg

Treg

Treg

Treg

2998400

2998400 Breg

Th17Th22



Abbreviations












References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)

0

10

20

30

40

50

60

lt0001 lt0001 lt0001

0008 0023

TGF-1205731

imm

unor

eact

ive c

ells

()





(b)


(c)

0

10

20

30

40

50

60

lt0001

lt0001 lt0001 lt0001

lt0001

IL-4

imm

unor

eact

ive c

ells

()





(d)


times320

(e)

0

10

20

30

40

50

60

lt0001 0015

lt0001 lt0001 lt0001

IL-1

3 im

mun

orea

ctiv

e cel

ls (

)





(f)




(a)

0

10

20

30

40

50

60

CD25

Fox

p3 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0001

0001





(b)


(c)

0

10

20

30

40

50

60

CD20

IL-1

0 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0006

0049





(d)


times320

(e)

0

10

20

30

40

50

60

FAS

imm

unor

eact

ive c

ells

()

lt0001 lt0001 lt0001

0008

0006





(f)







Median 14 61 57 113




Median 17 41 64 100




Median 16 25 47 42




Median 17 26 32 37




Median 46 42 61 108









Th22

()

0

2

4

6

8

10

12

14

16

lt0001 lt0001 lt0001

lt0001

lt0001

(a)Th

17 (

)

lt0001 lt0001 lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

0038

(b)

Th2

()

lt0001 0002

lt0001

0

2

4

6

8

10

12

14

16

0047

0035

(c)

Th1

()

lt0001 lt0001

0032

0

2

4

6

8

10

12

14

16

(d)

Treg

s (

)lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

MeanMedian




(e)








200x

(a)

Ma2Ta

Positive control

(b)



















times200

(a)

RT-PCR for HSV-1


times200

(b)


times320

(c)





Th1 TregsBregs

pDCregs


autoantibodies etc

Apoptosis ofneurons





Th22Th17TregsBregs

pDCregs






FibrosisMMPTIMP-1

Th1Th2

TregsBregspDCs

Th22

Th1ThThBreg

pDCTh2Th2

pDC

gggggggggggggg

Th2

Th2Th1

pDCTh2

Th1

Th17Th22

Th17

Breg

B

Th1ThThBreg

pDC Th1Th1Th1Th11

Th1Th2

Achalasia 8

7

6

5

4

3

1

2

4

TGF-1205731IL-4IL-13

(HLA-DQ1205731 DQ1205721 etc)

Treg

Treg

Treg

Treg

Treg

Treg

2998400

2998400 Breg

Th17Th22



Abbreviations












References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)

0

10

20

30

40

50

60

CD25

Fox

p3 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0001

0001





(b)


(c)

0

10

20

30

40

50

60

CD20

IL-1

0 im

mun

orea

ctiv

e cel

ls (

)

lt0001 lt0001 lt0001

0006

0049





(d)


times320

(e)

0

10

20

30

40

50

60

FAS

imm

unor

eact

ive c

ells

()

lt0001 lt0001 lt0001

0008

0006





(f)







Median 14 61 57 113




Median 17 41 64 100




Median 16 25 47 42




Median 17 26 32 37




Median 46 42 61 108









Th22

()

0

2

4

6

8

10

12

14

16

lt0001 lt0001 lt0001

lt0001

lt0001

(a)Th

17 (

)

lt0001 lt0001 lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

0038

(b)

Th2

()

lt0001 0002

lt0001

0

2

4

6

8

10

12

14

16

0047

0035

(c)

Th1

()

lt0001 lt0001

0032

0

2

4

6

8

10

12

14

16

(d)

Treg

s (

)lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

MeanMedian




(e)








200x

(a)

Ma2Ta

Positive control

(b)



















times200

(a)

RT-PCR for HSV-1


times200

(b)


times320

(c)





Th1 TregsBregs

pDCregs


autoantibodies etc

Apoptosis ofneurons





Th22Th17TregsBregs

pDCregs






FibrosisMMPTIMP-1

Th1Th2

TregsBregspDCs

Th22

Th1ThThBreg

pDCTh2Th2

pDC

gggggggggggggg

Th2

Th2Th1

pDCTh2

Th1

Th17Th22

Th17

Breg

B

Th1ThThBreg

pDC Th1Th1Th1Th11

Th1Th2

Achalasia 8

7

6

5

4

3

1

2

4

TGF-1205731IL-4IL-13

(HLA-DQ1205731 DQ1205721 etc)

Treg

Treg

Treg

Treg

Treg

Treg

2998400

2998400 Breg

Th17Th22



Abbreviations












References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Median 14 61 57 113




Median 17 41 64 100




Median 16 25 47 42




Median 17 26 32 37




Median 46 42 61 108









Th22

()

0

2

4

6

8

10

12

14

16

lt0001 lt0001 lt0001

lt0001

lt0001

(a)Th

17 (

)

lt0001 lt0001 lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

0038

(b)

Th2

()

lt0001 0002

lt0001

0

2

4

6

8

10

12

14

16

0047

0035

(c)

Th1

()

lt0001 lt0001

0032

0

2

4

6

8

10

12

14

16

(d)

Treg

s (

)lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

MeanMedian




(e)








200x

(a)

Ma2Ta

Positive control

(b)



















times200

(a)

RT-PCR for HSV-1


times200

(b)


times320

(c)





Th1 TregsBregs

pDCregs


autoantibodies etc

Apoptosis ofneurons





Th22Th17TregsBregs

pDCregs






FibrosisMMPTIMP-1

Th1Th2

TregsBregspDCs

Th22

Th1ThThBreg

pDCTh2Th2

pDC

gggggggggggggg

Th2

Th2Th1

pDCTh2

Th1

Th17Th22

Th17

Breg

B

Th1ThThBreg

pDC Th1Th1Th1Th11

Th1Th2

Achalasia 8

7

6

5

4

3

1

2

4

TGF-1205731IL-4IL-13

(HLA-DQ1205731 DQ1205721 etc)

Treg

Treg

Treg

Treg

Treg

Treg

2998400

2998400 Breg

Th17Th22



Abbreviations












References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Th22

()

0

2

4

6

8

10

12

14

16

lt0001 lt0001 lt0001

lt0001

lt0001

(a)Th

17 (

)

lt0001 lt0001 lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

0038

(b)

Th2

()

lt0001 0002

lt0001

0

2

4

6

8

10

12

14

16

0047

0035

(c)

Th1

()

lt0001 lt0001

0032

0

2

4

6

8

10

12

14

16

(d)

Treg

s (

)lt0001

lt0001

lt0001

0

2

4

6

8

10

12

14

16

MeanMedian




(e)








200x

(a)

Ma2Ta

Positive control

(b)



















times200

(a)

RT-PCR for HSV-1


times200

(b)


times320

(c)





Th1 TregsBregs

pDCregs


autoantibodies etc

Apoptosis ofneurons





Th22Th17TregsBregs

pDCregs






FibrosisMMPTIMP-1

Th1Th2

TregsBregspDCs

Th22

Th1ThThBreg

pDCTh2Th2

pDC

gggggggggggggg

Th2

Th2Th1

pDCTh2

Th1

Th17Th22

Th17

Breg

B

Th1ThThBreg

pDC Th1Th1Th1Th11

Th1Th2

Achalasia 8

7

6

5

4

3

1

2

4

TGF-1205731IL-4IL-13

(HLA-DQ1205731 DQ1205721 etc)

Treg

Treg

Treg

Treg

Treg

Treg

2998400

2998400 Breg

Th17Th22



Abbreviations












References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















200x

(a)

Ma2Ta

Positive control

(b)



















times200

(a)

RT-PCR for HSV-1


times200

(b)


times320

(c)





Th1 TregsBregs

pDCregs


autoantibodies etc

Apoptosis ofneurons





Th22Th17TregsBregs

pDCregs






FibrosisMMPTIMP-1

Th1Th2

TregsBregspDCs

Th22

Th1ThThBreg

pDCTh2Th2

pDC

gggggggggggggg

Th2

Th2Th1

pDCTh2

Th1

Th17Th22

Th17

Breg

B

Th1ThThBreg

pDC Th1Th1Th1Th11

Th1Th2

Achalasia 8

7

6

5

4

3

1

2

4

TGF-1205731IL-4IL-13

(HLA-DQ1205731 DQ1205721 etc)

Treg

Treg

Treg

Treg

Treg

Treg

2998400

2998400 Breg

Th17Th22



Abbreviations












References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















times200

(a)

RT-PCR for HSV-1


times200

(b)


times320

(c)





Th1 TregsBregs

pDCregs


autoantibodies etc

Apoptosis ofneurons





Th22Th17TregsBregs

pDCregs






FibrosisMMPTIMP-1

Th1Th2

TregsBregspDCs

Th22

Th1ThThBreg

pDCTh2Th2

pDC

gggggggggggggg

Th2

Th2Th1

pDCTh2

Th1

Th17Th22

Th17

Breg

B

Th1ThThBreg

pDC Th1Th1Th1Th11

Th1Th2

Achalasia 8

7

6

5

4

3

1

2

4

TGF-1205731IL-4IL-13

(HLA-DQ1205731 DQ1205721 etc)

Treg

Treg

Treg

Treg

Treg

Treg

2998400

2998400 Breg

Th17Th22



Abbreviations












References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Th1 TregsBregs

pDCregs


autoantibodies etc

Apoptosis ofneurons





Th22Th17TregsBregs

pDCregs






FibrosisMMPTIMP-1

Th1Th2

TregsBregspDCs

Th22

Th1ThThBreg

pDCTh2Th2

pDC

gggggggggggggg

Th2

Th2Th1

pDCTh2

Th1

Th17Th22

Th17

Breg

B

Th1ThThBreg

pDC Th1Th1Th1Th11

Th1Th2

Achalasia 8

7

6

5

4

3

1

2

4

TGF-1205731IL-4IL-13

(HLA-DQ1205731 DQ1205721 etc)

Treg

Treg

Treg

Treg

Treg

Treg

2998400

2998400 Breg

Th17Th22



Abbreviations












References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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