Brain-Reactive Igg Correlates with Autoimmunity in Mothers of a Child with an Autism Spectrum Disorder

IMMEDIATE COMMUNICATION Brain-reactive IgG correlates with autoimmunity in mothers of a child with an autism spectrum disorder

L Brimberg1, A Sadiq1, PK Gregersen2 and B Diamond1

It is believed that in utero environmental factors contribute to autism spectrum disorder (ASD). The goal of this study was to demonstrate, using the largest cohort reported so far, that mothers of an ASD child have an elevated frequency of anti-brain antibodies and to assess whether brain reactivity is associated with an autoimmune diathesis of the mother. We screened plasma of 2431 mothers of an ASD child from Simon Simplex Collection and plasma of 653 unselected women of child-bearing age for anti-brain antibodies using immunohistology on mouse brain. Positive and negative plasma from mothers with an ASD child were analyzed for anti-nuclear antibodies and for autoimmune disorders. Mothers of an ASD child were four times more likely to harbor anti-brain antibodies than unselected women of child-bearing age (10.5 vs 2.6%). A second cohort from The Autism Genetic Resource Exchange with multiplex families displayed an 8.8% prevalence of anti-brain antibodies in the mothers of these families. Fifty-three percent of these mothers with anti-brain antibodies also exhibited anti-nuclear autoantibodies compared with 13.4% of mothers of an ASD child without anti-brain antibodies and 15% of control women of child-bearing age. The analysis of ASD mothers with brain-reactive antibodies also revealed an increased prevalence of autoimmune diseases, especially rheumatoid arthritis and systemic lupus erythematosus. This study provides robust evidence that brain-reactive antibodies are increased in mothers of an ASD child and may be associated with autoimmunity. The current study serves as a benchmark and justification for studying the potential pathogenicity of these antibodies on the developing brain. The detailed characterization of the specificity of these antibodies will provide practical benefits for the management and prevention of this disorder.

Molecular Psychiatry (2013) 18, 1171–1177; doi:10.1038/mp.2013.101; published online 20 August 2013 Keywords: anti-brain antibodies; ASD; autoimmunity; maternal

INTRODUCTION child with ASD. Other studies have addressed the potential risk of Autism spectrum disorder (ASD) is a complex neurodevelopmental exposure to maternal antibody in utero. Specifically, several disorder with poorly defined and complex etiology, characterized investigators have identified the presence of antibodies that bind by a broad range of symptoms in the domains of socialization and to human fetal brain tissue in mothers with an ASD offspring.8,9 communication and by restricted behavioral patterns.1Over the When these antibodies were administered to gestating mice or past decades, a substantial increase in the prevalence of ASD has monkeys, the offspring exhibited abnormal behavior (reviewed by been reported with a current estimation of over 1% incidence in Enstrom et al.6). In an early study, serum-containing antibodies 2 the general population. The need for early diagnosis and reactive with neuronal antigens from a mother of an ASD child prevention is pressing. Although the rapid increase in incidence was passively administered to pregnant mice. The offspring were may be in part attributable to better diagnosis and public observed to have deficits in social behavior and motor skills and awareness, environmental causes may also contribute. cerebellar abnormalities on histopathology.10 In a subsequent It has become apparent that brain-reactive antibodies can study, IgG isolated from pooled sera of mothers of a child with contribute to human pathobiology. Some well-characterized ASD were administrated to pregnant mice. The in utero exposed examples of antibody-mediated neurological disease are neuro- offspring had increased activity at a young age as well as anxiety- myelitis optica, paraneoplastic syndromes and neuropsychiatric like behavior, alterations in sociability and increased startle lupus for which pathogenic antibodies that target antigens in the 11 brain have been identified (for a review see the study by Diamond following acoustic stimuli in adulthood. In addition to these et al.3). Although much less well-characterized, anti-brain studies in rodents, IgG pooled from sera of mothers of a child with antibodies have also been implicated in the pathogenesis of a ASD were administrated to pregnant rhesus monkeys. The variety of childhood neuropsychiatric syndromes such as offspring were observed to have social deficits, increased motor obsessive compulsive disorder,4 Tourette’s syndrome5 and ASD.6 activity and increased stereotypic behaviors compared with Several studies have linked maternal infections or inflammation monkeys born of mothers given control IgG pooled from during pregnancy to the development of ASD in the offspring mothers of a normally developing child.12 The results from these (reviewed by Patterson7), suggesting that activation of the studies suggest that maternal antibodies targeting brain antigens maternal immune system might lead to an increased risk of a can alter neurodevelopment in the fetus.

1Center for Autoimmune and Musculoskeletal Diseases, Manhasset, NY, USA and 2Center for Genomics and Human Genetics, The Feinstein Institute for Medical Research (FIMR), Manhasset, NY, USA. Correspondence: Dr B Diamond, Center for Autoimmune and Musculoskeletal Diseases, The Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, NY 11030, USA. E-mail: [email protected] Received 8 April 2013; revised 3 July 2013; accepted 10 July 2013; published online 20 August 2013 Maternal autoimmunity and autism spectrum disorder L Brimberg et al 1172 A number of epidemiological studies have found an association subjects (that is, mother of an ASD child; control). A third reviewer, also between a maternal history of autoimmune disease and increased blind to the status of the subjects, assessed random samples (B10%). risk of having a child with ASD. Increased risk of ASD in the child was found if the mother was diagnosed with psoriasis,13 Anti-nuclear antibody staining. Anti-nuclear antibodies were assayed in rheumatoid arthritis or celiac disease.14 With a substantial human plasma utilizing the Anti-Nuclear Antibody test system (MBL-Bion, increase in the prevalence of ASD, as has also been observed Des Plains, IL, USA). Plasma was diluted 1:100 in PBS. Staining was performed 15 according to the manufacturer’s instructions and visualized with OpenLab for most autoimmune diseases over the past four decades, we software on an Axio-Plan2 microscope (Zeiss). hypothesize that production of brain-reactive antibodies may be associated with features of autoimmunity. The goal of this study was to provide evidence, using the largest Western blot cohort reported so far, that mothers of an ASD child have Whole- and membrane human fetal brain lysates, 10 ug per lane an elevated frequency of anti-brain antibodies and to assess (Invitrogen), were separated by SDS–PAGE electrophoresis using NuPAGE whether brain reactivity is associated with an autoimmune (Invitrogen) and transferred to polyvinylidene ﬂuoride membranes. Membranes were incubated with plasma (1:100 in PBS solution with diathesis of the mother. Tween 20) followed by incubation with anti-human IR Dye 800CW and The implication of this work could lead, in the future, to visualized using an Odyssey Infrared Imaging System (LI-COR Biosciences, prevention of some proportion of ASD. Lincoln, NE, USA).

Statistical analysis METHODS Two-sample Student’s t-tests were used to compare means of two Research subjects independent groups. To analyze categorical data, a w2-test for indepen- Plasma from mothers with an ASD child was obtained from the Simons dence was used. When expected sample size was smaller than 5, the Simplex Collection (SSC, http://sfari.org/resources/simons-simplex- Fisher’s exact test was used. Values were considered significant for P 0.05. 16 o collection) and the Autism Genetic Resource Exchange (AGRE, http:// For multiple comparisons, the Keppel multiple comparisons correction was agre.autismspeaks.org). Details on recruitment of families, consent and used. protection of the privacy of participants, evaluation and basic description of the cohort can be found for the SSC by Fischbach and Lord16 and for AGRE 17 by Geschwind et al. RESULTS Control plasma from women of child-bearing age were obtained from the North-Shore-LIJ health system clinical laboratory, participants in a To test whether mothers of an ASD child are more likely to harbor registry at the Feinstein Institute for Medical Research (http://www.gapre- anti-brain antibodies than unselected women of child-bearing gistry.org) and a previously characterized adult cohort enrolled in the age, we assessed plasma reactivity to mouse brain tissue. metropolitan New York area, as described by Mitchell et al.18 No other Intensity was determined to be an absolute negative when no selection criteria were applied to these control subjects, and no other staining was visualized, positive when strong staining was information was obtained. visualized and indeterminate for all other cases (Supplementary Plasma of women with rheumatoid arthritis were derived from subjects Figure 1). There was no disagreement between the observers on enrolled in the the National Data Bank for Rheumatic Diseases, a positive and negative plasma. longitudinal cohort of rheumatoid arthritis patients recruited across the United States19 that has been included in previous genetic studies of The study was performed on 2431 mothers of an ASD child 20 obtained from the SSC and 653 controls obtained from local New rheumatoid arthritis. A full description of this cohort including 18 demographic and clinical variables can be found in the study by Wolfe York cohorts, the Feinstein Institute (www.gapregistry.com) and et al.19 the North-Shore University Hospital laboratories. Table 1 Individuals provided informed consent through the appropriate institu- describes, by each source of subjects, race, ethnicity and the tional review board. age of the subject when blood was drawn. Mothers of an ASD child are nearly four times more likely to Immunohistology harbor anti-brain antibodies than unselected women of Preparation of adult mouse tissue sections. Twelve-week-old unmanipu- child-bearing age (Po0.00001). In total, 10.7% of the plasma of lated C57BL/6 mice (Jackson Laboratories, Bar Harbor, MA, USA) were mothers of an ASD child (260/2431) displayed strong reactivity anesthetized with isoflurane before perfusion. Brains were perfused with 4% to mouse brain antigens compared with 2.6% plasma of paraformaldehyde, following replacement of circulation with heparanized unselected women of child-bearing age (17/653). Only 28% preperfusion buffer. Brains were removed, postfixed over-night and plasma of mothers of an ASD child (682/2431) showed no binding infiltrated with 30% sucrose 48 h at 4 1C. Brains were next frozen in freezing compared with 64.7% plasma of unselected women of child- medium (O.C.T. compound, Sakura, San Francisco, CA, USA) on dry ice and bearing age (423/653). The remaining women showed indetermi- 1 stored at 80 C until sectioning. Transverse sections of 12 mm were mounted nate binding. on gelatin-coated slides and stored at 80 1C until use. Procedures were 2 approved by the Institutional Animal Care and Use Committee. w -Test yielded no difference in the presence of anti-brain antibodies between the different sources of controls (see Preparation of embryonic brain tissue sections. The procedure Supplementary Table 1, for detailed analysis); thus, they were was done as described in Bond et al.21 with slight modifications. Brains analyzed as a single cohort in subsequent analyses. were sectioned as described for the adult mouse brain preparation. The presence of antibodies to brain tissue within a subgroup of the mothers of an ASD child could not be attributed to Immunostaining for anti-brain antibodies. To assess IgG reactivity to the confounding factors such as the time elapsed from giving birth brain, 12-mm sections were air dried, rinsed twice for 5 min with phosphate to the time blood was collected (Table 2), the age when she gave buffered saline (PBS) containing 0.1% Tween-20. Following blocking for 1 h birth (Table 2) or to any reported complication or illnesses during with PBS containing 10% heat-inactivated normal goat serum in 0.2% pregnancy including fever, infections, allergies and diabetes Triton X100 in at room temperature, sections were incubated for 1 h with (Supplementary Table 4). No differences in demographic, socio- 1:50 human plasma diluted in PBS with 3% heat-inactivated normal goat economic (Supplementary Table 2) and psychiatry evaluation serum in 0.2% Triton X100. After washing in PBS/Tween, binding of plasma to the brain was detected using Alexa 488 goat anti-human IgG (Supplementary Table 3) were found between mothers of an ASD (Invitrogen, Grand Island, NY, USA) and visualized with OpenLab software child with or without anti-brain antibodies. on an Axio-Plan2 microscope (Zeiss, Thornwood, NY, USA). In order to confirm that the current observation was not Intensity and localization of reactivity was determined by two restricted to subjects enrolled in the SSC, we analyzed additional independent observers. One observer was blind to the status of the 318 plasma of mothers of an ASD child from AGRE (demographic

Molecular Psychiatry (2013), 1171 – 1177 & 2013 Macmillan Publishers Limited Maternal autoimmunity and autism spectrum disorder L Brimberg et al 1173 data are described in Supplementary Table 5). In all, 8.8% of the Immunohistology of adult mouse brain was corroborated plasma of mothers of an ASD child (28/318) displayed strong by immunohistology of mouse fetal brain (data not shown) reactivity to mouse brain antigens. Only 22.6% plasma of mothers and western blots of human fetal brain lysates. Distinct of an ASD child (72/318) showed no binding. The remaining bands were commonly bound by IgG from positive plasma, women showed indeterminate binding. w2-Analysis yielded no although there was also binding on western blot by some difference between sources (AGRE and SSC, P40.5). plasma that were negative by immunohistology (Supplementary Staining patterns from antibody-positive woman localized to Figure 2). distinct regions of the adult mouse brain (Figure 1). Generally, We hypothesized that brain reactivity would be associated with binding to neurons in the frontal cortex, hippocampus and the an autoimmune predisposition; we, therefore, tested for the cerebellum was evident. In only 5 of 288 cases was there clear presence of anti-nuclear antibodies, which are commonly present binding to glial cells in addition to neurons. in individuals with many autoimmune diseases.22 Reactivity was assessed as positive, negative or indeterminate (Supplementary Figure 3). Table 1. Demographic of subjects As can be seen in Table 3, mothers of an ASD child who were positive for anti-brain antibodies were significantly more likely to Race (%) harbor anti-nuclear autoantibodies than mothers of an ASD child or unselected women of child-bearing age who lacked anti-brain antibodies (w2-test, P 0.0001). There was no significant difference African– o White American Asian Other between mothers of an ASD child with no anti-brain antibodies and controls. In total, 53% (152/284) of mothers of an ASD SSC 80 3.9 5 11.1 child with anti-brain antibodies also exhibited anti-nuclear NYC population cohort 100 0 0 0 autoantibodies compared with 13.4% (99/738) of mothers GAP 76 3.3 8.9 11.8 of an ASD child without anti-brain antibodies and 15% (52/345) North-Shore-LIJ 70 13 6 11 of unselected women of child-bearing age. Only 23.2% (66/284) laboratories of mothers of an ASD child with anti-brain antibodies showed no binding compared with 54.6% (403/738) of mothers of Ethnicity (%) an ASD child without anti-brain antibodies and 62.3% (215/345) of unselected women of child-bearing age. The remaining Hispanic Non-Hispanic subjects showed indeterminate binding. These data are consistent with a predisposition to more generalized autoimmunity SSC 5 95 in some mothers with anti-brain antibodies who have a child NYC population cohort 0 100 with ASD. GAP 7 93 Next, we used the self-reported data available from the SSC to North-Shore-LIJ laboratories 5 95 analyze whether autoimmune diseases were more common in mothers of an ASD child with anti-brain antibodies compared with Age at the time blood was drawn mothers of an ASD child who lack anti-brain antibodies. Data were available only for subjects from the SSC and included the Mean s.d. Min Max following diseases: juvenile inflammatory arthritis, rheumatoid arthritis, systemic lupus erythematosus, Hashimoto’s thyroiditis, SSC 40.28 5.73 22 58 celiac disease, type 1 diabetes, psoriasis, multiple sclerosis and NYC population cohort 43 3.42 37 49 adrenal deficiency. The number and percentage of cases for each GAP 38.8 9.6 19 50 disease are presented in Supplementary Table 6. The analysis of North-Shore-LIJ laboratories 36.17 8.14 18 50 ASD mothers with anti-brain antibodies revealed an increased Abbreviations: ASD, autism spectrum disorder; GAP, genotype and incidence of autoimmune diseases especially rheumatoid arthritis, phenotype; LIJ, Long Island Jewish; NYC, New York City; SSC, Simons and systemic lupus erythematosus (Table 4). Simplex Collection. Finally, to ask whether autoimmunity predisposes to production of anti-brain antibodies, we determined whether women with

Table 2. Characterization of mothers of an ASD child

Mothers of an ASD child

Anti-brain antibodies Anti-brain antibodies negative (N ¼ 644) positive (N ¼ 238) P-valuea

Age at the time the blood was drawn Mean s.d. Min Max Mean s.d. Min Max 39.9 5.74 22 57 36.61 6.05 23 56 NS Years elapsed between birth of the child and the time Mean s.d. Min Max Mean s.d. Min Max blood was drawn 8.87 3.45 4 18 9.09 3.71 3 19 NS Age at the time of giving birth Mean s.d. Min Max Mean s.d. Min Max 31.01 5.021 16 45 31.5 5.36 18 44 NS Has the subject had pregnancies that did not end in Yes No Yes No live birth? (%) 31 69 30 70 NS Abbreviations: ASD, autism spectrum disorder; NS, not signiﬁcant. aStudent’s t-test or w2-test for categorical data, all P-values 40.12.

& 2013 Macmillan Publishers Limited Molecular Psychiatry (2013), 1171 – 1177 Maternal autoimmunity and autism spectrum disorder L Brimberg et al 1174 Unselected women of child bearing age Mother of an ASD child a x5 x5 x40 Hippocampus

b x5 x5 x40 Cerebellum

c x5 x40 x5 Frontal Cortex

Figure 1. Representative plasma showing reactivity of plasma of an unselected woman of child-bearing age (ﬁrst panel) and mother of an autism spectrum disorder (ASD) child to mouse adult brain (second and third panel). (a–c) Transverse tissue sections were incubated with plasma of mother of an ASD child or control. Anti-brain IgG was detected using Alexa 488 anti-human IgG. As can be seen, plasma of mother of an ASD child primarily labeled the (a) hippocampus, (b) cerebellum and (c) frontal cortex 5, 40 state the magniﬁcation.

Table 3. Mothers with anti-brain antibodies and a child with ASD are more likely harbor for anti-nuclear antibodies

Anti-nuclear antibodies

Subjects Source Number of subjects Negative Positive

Mothers of an ASD child anti-brain IgG positive SSC 256 22.65% (n ¼ 56) 53.5% (n ¼ 137) AGRE 28 35% (n ¼ 10) 53% (n ¼ 15) Mothers of an ASD child anti-brain IgG negative SSC 667 55.5% (n ¼ 357) 12.7% (n ¼ 85) AGRE 71 64.7% (n ¼ 46) 19.7% (n ¼ 14) Unselected women of child-bearing age aCombined 345 62.3% (n ¼ 215) 15% (n ¼ 52) anti-brain IgG negative Abbreviations: AGRE, Autism Genetic Resource Exchange; ASD, autism spectrum disorder; SSC, Simons Simplex Collection. w2-Analysis yielded no signiﬁcant difference between the two cohorts for mothers of an ASD child positive for anti-brain antibodies or mothers of an ASD child negative for anti-brain antibodies, or controls. The remaining subjects in each cohort showed indeterminate binding (see Supplementary Figure 3). aControl includes subjects for the NYC population study, GAP and North-Shore-lij laboratories.

rheumatoid arthritis (n ¼ 363) were more likely to have anti-brain DISCUSSION antibodies compared with the control women. Interestingly, we The passage of maternal antibodies across the placenta is a well- found that women with rheumatoid arthritis are as likely to have known mechanism for fetal immune protection. In the fetus, the anti-brain antibodies as mothers of an ASD child (rheumatoid blood–brain barrier is not fully formed, making the developing arthritis positive, 13.5% (49/362); negative 32.3% (117/362); brain vulnerable to blood-borne substances.23 In utero exposure indeterminate 54.1% (196/362)). Supplementary Table 7 describes to maternal anti-brain antibodies has been posited as an race and ethnicity, age of disease onset and the age when blood important potential trigger for abnormal brain development.3 was drawn of women with rheumatoid arthritis. None of these Growing evidence suggests that maternal antibodies can target variables could account for the difference in the presence of anti- the fetal brain, as maternal antibody can penetrate fetal brain brain antibodies. tissue, which is not protected by a fully functional blood–brain

Molecular Psychiatry (2013), 1171 – 1177 & 2013 Macmillan Publishers Limited Maternal autoimmunity and autism spectrum disorder L Brimberg et al 1175 Table 4. Autoimmune diseases are more common in mothers with anti-brain antibodies and a child with ASD

Mothers of an ASD child

Disorder Anti-brain antibody negative (n ¼ 622) Anti-brain antibody positive (n ¼ 233) w2- value P-valuea

Rheumatoid arthritis n ¼ 9 1.45% n ¼ 9 4% 4.82 0.028 Systemic lupus erythematosus n ¼ 1 0.16% n ¼ 5 2.22% 9.58 0.0068 Total number of autoimmune diseases n ¼ 55 8.8% n ¼ 43 18.4% 15.4 0.0005 Abbreviation: ASD, autism spectrum disorder. Table 4 presents the significant results from the data that was available for subjects from SSC. For each disease the number of positive cases and the percentage that these cases represent is shown. The full analysis (Supplementary Table 4) includes the following diseases: Juvenile inflammatory arthritis, rheumatoid arthritis, systemic lupus erythematosus (SLE), Hashimoto’s thyroiditis, celiac disease, type 1 diabetes, psoriasis, multiple sclerosis, adrenal insufficiency. aKeppel multiple comparison correction, Po0.035. barrier. For example, anti-DNA, anti-N-methyl-D aspartate recep- fetal brain as well, but it was not possible to localize regions of tor, cross-reactive antibodies specifically present in women with immunoreactivity in fetal brain. Although we were also able to systemic lupus erythematosus, have been shown in mouse models demonstrate bands on western blot, the bands identified were to be neurotoxic to the developing brain.24 More recently, it has variable using a limited number of seropositive and seronegative been shown that these antibodies have differential effects on plasma making it difficult to characterize plasma as positive or male and female fetuses, causing cognitive impairments in the negative. These data, did however, show that binding to tissue former and fetal death in the latter.25 These antibodies affect the antigens was not species-specific. Reactivity across species is also adult mouse if, and only if, there is a compromise to blood–brain consistent with the studies mentioned above showing that anti- barrier integrity.24 brain serum or IgG from mothers of an ASD child given to Limited reports have consistently shown that when anti-brain pregnant mice or monkeys cause cognitive and behavioral antibodies from mothers of an ASD child were administrated abnormalities in the offspring.11,12 It has also been shown that to pregnant mice or pregnant monkeys, the offspring many human autoantibodies target highly conserved region of exhibit behavioral alterations analogous to those seen in ASD the autoantigen and therefore reacts with antigen present in children.11,12 multiple species.29,30 Using the largest cohort reported so far, we have provided We demonstrated that anti-nuclear antibodies and autoimmune evidence that mothers of an ASD child are significantly more likely disease are increased in mothers with anti-brain antibodies and to harbor anti-brain antibodies than age-matched unselected with an ASD child. The possibility of autoimmune mechanisms women. This observation was true with respect to women in the being a contributing factor in ASD has been entertained as early SSC and with respect to women in the AGRE collection. studies suggested that individuals with ASD have a family history Our results are also consistent with previous relatively small of autoimmune disease.14,31 A recent large study examined studies.8,9,26 autoimmune disorders in women with over 600 000 births and The majority of previous reports have used western blotting of showed that women with either rheumatoid arthritis or celiac brain antigens as a primary screening tool. The specific brain disease have an increased risk of having a child with ASD.14 antigens detected in these studies have not yet been identified, Interestingly, there was no increased incidence of rheumatoid although some studies have identified 39 and 73 kDa bands that arthritis in fathers of a child with ASD, consistent with the are often seen on western blots of brain lysates.8,9,26 In this hypothesis that the in utero experience may contribute to ASD in study, we have screened for anti-brain antibodies by histology of the offspring. adult mouse brain sections. We, as others, have not identified a Our findings of increased autoimmune disease and specifi- specific antigenic target; yet, the use of an immunohistological cally rheumatoid arthritis and systemic lupus erythematosus assay allowed us to identify the brain regions exhibiting are consistent with the hypothesis that autoimmune diseases the highest reactivity with maternal IgG, including the frontal may confer risk for ASD in the offspring of mothers with these cortex, hippocampus and cerebellum. These regions have been disorders. implicated in ASD through imaging and post mortem studies;27 We have shown that an autoimmune diathesis such as is cytoarchitectural organizational abnormalities of the cerebral present in women with rheumatoid arthritis predisposes to the cortex, limbic area, cerebellum, as well as other subcortical production of anti-brain antibodies in a similar proportion to what structures have been documented. In particular, studies have we reported for mothers with an ASD child. Plasma of women with demonstrated neuronal abnormalities such as loss and atrophy rheumatoid arthritis showed similar staining patterns as plasma of of Purkinje cells, abnormal development of neurons in the mothers with an ASD child. Future studies will need to determine hippocampus and changes in neuronal size and number in the whether common antigens are involved. It is interesting to cerebellum and midbrain.27,28 The immunostaining patterns we postulate that production of anti-brain antibodies may be the observed were almost exclusively neuronal and confined mechanism for the reported associations between autoimmune to regions that have consistently been shown to be involved in disease and ASD. We have shown that mothers of an ASD child ASD, thus strongly supporting the physiological relevance of who were positive for anti-brain antibodies had more self- the antibodies. Their specificity and functional role remain to be reported autoimmune conditions than mothers of an ASD child determined. with no anti-brain antibodies. We also found that anti-nuclear The utilization of immunohistology of adult mouse brain antibodies, a common marker of both subclinical autoimmunity as allowed us to screen relatively quickly the large number of well as clinical autoimmune disease, are more frequent in mothers samples reported in this study as well as to broadly identify of an ASD child who harbor anti-brain antibodies. the brain regions targeted by antibody. In order to confirm the A large number of genetic risk variants have been identified relevance of the findings to mouse fetal brain and human, we with autoimmune disorders,32 and we have considered the performed immunohistology on fetal mouse brain sections as possibility that these variants may also predispose to the deve- western blotting using commercially available human fetal brain lopment of anti-brain antibodies in mothers of an ASD child. Our lysates. Without expectation, plasma binding to adult brain bound preliminary studies of mothers in the SSC for whom genetic data

& 2013 Macmillan Publishers Limited Molecular Psychiatry (2013), 1171 – 1177 Maternal autoimmunity and autism spectrum disorder L Brimberg et al 1176 were available have not revealed strong evidence of associa- antibodies may lead to autism in the offspring. Bauman et al.37 tions with established autoimmune loci. However, our sample demonstrated the pathogenic potential of antibodies directed size is small and large cohorts will be required to perform against specific fetal brain proteins in rhesus monkeys. a definitive analysis of autoimmune risk alleles in mothers of a Braunschweig et al.38 described a panel of six proteins that child with ASD. might be recognized by those anti-brain antibodies. In a preliminary analysis on a subset of ASD subjects, we did not find an association between the presence of maternal anti-brain antibodies and the age of diagnosis of the child, severity of CONFLICT OF INTEREST disease, IQ or distinct behavioral patterns in the child (data not The authors declare no conflict of interest. shown). We believe that this may reflect the heterogeneity of the antigenic specificity of anti-brain antibodies. One previous report, however, did demonstrate a correlation between maternal anti- ACKNOWLEDGEMENTS body reactivity with specific bands on a western blot of brain We thank Dr Marta Benedetti at the Simons Foundation and William Jensen at lysates and irritability and communication impairments in the 33 Prometheus Research for assistance with obtaining and analyzing clinical data affected offspring. Once distinct antibody specificities are on the Simons collection. We also thank Matt State for access to genetic data on identified, we will repeat the analysis if phenotypes of children mothers in the Simons Collection; Bradley and Jennifer Marsh for seed funding for with ASD relate to target antigens. this project. This work was supported by Simons Foundation, Autism Speaks. We evaluated antibodies from blood that was obtained from the mother years after pregnancy. Our assumption is that these antibodies reflect a chronic immune state of the mother, REFERENCES in which the abnormal serology persists for years, and thus was 1 Mahjouri S, Lord CE. What the DSM-5 portends for research, diagnosis, present at the time of pregnancy. Autoantibodies have and treatment of autism spectrum disorders. 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Supplementary Information accompanies the paper on the Molecular Psychiatry website (http://www.nature.com/mp)

& 2013 Macmillan Publishers Limited Molecular Psychiatry (2013), 1171 – 1177 Medical Hypotheses 77 (2011) 494–504

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Medical Hypotheses

journal homepage: www.elsevier.com/locate/mehy

Reconstitution of the human biome as the most reasonable solution for epidemics of allergic and autoimmune diseases ⇑ Staci D. Bilbo a, Gregory A. Wray b, Sarah E. Perkins c, William Parker d, a Department of Psychology and Neuroscience, Systems and Integrative Neuroscience Group, Duke University, Durham, NC 27710, United States b Department of Biology, Duke Institute for Genome Sciences and Policy, Duke University, Durham, NC 27710, United States c Cardiff School of Biosciences, Biomedical Sciences Building, Museum Avenue, Cardiff CF10 3AX, UK d Department of Surgery, Duke University Medical Center, Durham, NC 27710, United States article info abstract

Article history: A wide range of hyperimmune-associated diseases plague post-industrial society, with a prevalence and Received 30 March 2011 impact that is staggering. Strong evidence points towards a loss of helminths from the ecosystem of the Accepted 4 June 2011 human body (the human biome) as the most important factor in this epidemic. Helminths, intestinal worms which are largely eradicated by elements of post-industrial culture including toilets and water treatment facilities, have an otherwise ubiquitous presence in vertebrates, and have co-evolved with the immune system. Not only do helminths discourage allergic and autoimmune reactions by diverting the immune system away from these pathologic processes and stimulating host regulatory networks, helminths release a variety of factors which down-modulate the immune system. A comprehensive view of hyperimmune-related disease based on studies in immunology, parasitology, evolutionary biology, epidemiology, and neurobiology indicates that the effects of biome depletion may not yet be fully realized, and may have an unexpectedly broad impact on many areas of human biology, including cognition. For- tunately, colonization with helminths results in a cure of numerous autoimmune and allergic diseases in laboratory rodents, and clinical studies in humans have indicated their utility for treatment of both multiple sclerosis and inﬂammatory bowel disease. Based on these considerations, commitment of considerable resources toward understanding the effects of ‘‘biome depletion’’ and systematically evaluating the most effective approach toward biome reconstitution is strongly encouraged. Ó 2011 Elsevier Ltd. All rights reserved.

Introduction Epidemiology of hyperimmune-associated disease

The field of clinical immunology is faced with epidemics of a Epidemics of diseases associated with a hyperactive immune wide range of allergic, autoimmune, and other inflammation-asso- system occur much more frequently in countries that enjoy the ciated diseases with an impact that is staggering. These epidemics, widespread use of modern medical care, sanitation practices, and which appear to be products of post-industrial society, have water treatment technology [2–4]. These diseases can be associ- strongly resisted ever-increasing efforts focused on the develop- ated with an increased propensity for inflammation, and often in- ment of pharmaceuticals to combat the problem [1]. In this paper, volve pathologic immune reactivity directed against self and non- we examine evidence from the fields of evolutionary biology, ecol- self antigens, producing autoimmune and allergic diseases, respec- ogy, immunology, parasitology, neurobiology, and medicine for the tively. The wide range of these non-infectious conditions is strik- hypothesis that the only reasonable solution to these epidemics is ing, and includes lupus, multiple sclerosis, inflammatory bowel to reconstitute the ecosystem of the human body, the ‘‘human disease, hay fever, appendicitis, eczema, Graves’ disease, asthma, biome’’, with factors, especially helminths, that have been depleted and dangerously allergic reactions against antigens in common in post-industrial society. A range of arguments which might be food substances including nuts, milk and grains [5,6]. The potential mounted against this hypothesis are addressed, as well as the that some neurological disorders such as autism may also be implications for biomedical research and the field of clinical hyperimmune-associated [7] adds further to the milieu. The prev- immunology. alence of these diseases in post-industrial societies is staggering [6,8]. For example, more than one person in 1000 acquires lupus [9], approximately one in 600 acquires multiple sclerosis in some ⇑ Corresponding author. Address: Department of Surgery, Duke University Medical Center, Box 2605, Durham, NC 27710, United States. Tel.: +1 919 681 areas of the world [10], another one in 600 acquires type 1 diabetes 3886; fax: +1 919 681 7263. [11], one in 40 suffers from food allergies [12], approximately one E-mail address: [email protected] (W. Parker). in 20 acquires autoimmune thyroid disease [13], about one in 18

0306-9877/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2011.06.019 S.D. Bilbo et al. / Medical Hypotheses 77 (2011) 494–504 495 reports symptoms of asthma [14], about one in 13 acquires appendicitis [15], and approximately half of the population in the United States reacts against common allergens based on a skin test [16] and experiences symptoms such as a runny nose and itchy eyes that are consistent with allergic reactions [17]. The most widely supported view of the cause of this epidemic can be termed ‘‘The Biome Depletion Theory’’. This theory states that a profound depletion of components from the ecosystem of the human body, the ‘‘human biome’’, in post-industrial society has left our immune systems profoundly over-reactive, with a strong propensity to react against a wide range of non-pathogenic self and non-self antigens.

The human biome and the immune system: a single, functional unit

Fig. 1. Interdependence of the microbiome (microorganisms colonizing the human The Biome Depletion Theory is based on the intimate connection body), helminths colonizing the human body, and the immune system. Factors between the human immune system and those organisms that live having a strong effect on any one component potentially have both direct and in and on the human body. Studies in which animals are completely indirect effects on all other components. Each of these three major components is itself a complex and interdependent set of subcomponents, and potentially separated from all microorganisms have been instrumental in our interacts with other systems (e.g., endocrine, nervous, cardiovascular) in the understanding of the connection between the immune system human biome. and the rest of the human biome. Decades of experiments have reproducibly demonstrated that microbe-free animals, known as gnotobiotic or germ free animals, have an immune system which Coevolution of vertebrates with helminths is grossly undeveloped relative to the immune system of animals that are colonized with a normal bacterial flora [18–21]. The addi- The ancient coevolutionary history of the immune system with tion of bacteria to previously germ free animals results in restora- helminths provides insight into the entrenched nature of the im- tion of the immune system [21], and demonstrates the mune system-helminth connection. The evolutionary origin of dependence of the immune system on the microbial flora. Ongoing jawed fishes more than 400 million years ago marked not only work on the relationship between the microbial flora and the im- the appearance of immune systems containing all of the major mune system has led to the understanding that the immune system components found in humans, but also presumably provided suit- is better described as an interface with the environment more so able vertebrate hosts for flatworm parasites [30]. Although it re- than as a defensive wall against the environment. The immune sys- mains unknown when in evolutionary history helminths took up tem’s role as an environmental interface is illustrated by its support residence in the vertebrate gut, several lines of evidence point to- of mutualistic microorganisms that deter pathogenic microorgan- ward helminths residing in the guts of vertebrates more than isms, a process which potentially provides a more effective barrier 100 million years ago. For example, the observation that several to contagious disease than does anti-microbial measures carried clades (groups of related species) of invertebrates are composed out directly by the immune system [22]. entirely of species that are obligate associates of vertebrate guts Understanding the connection between the microorganisms suggests that gut colonization has been present for as long as these normally associated with our bodies and the immune system has clades have been in existence [31]. Thus, gut colonization probably garnered great interest, and knowledge in this field is expanding dates back to the origin of tapeworms (cestodes, with more than quickly. These human-associated microorganisms, collectively 1000 species), spiny-headed worms (acanthocephalans, with more known as the ‘‘microbiome’’, comprise an important part of the hu- than 1100 species), and several clades of flukes (trematodes) and man biome. The importance of this part of the human biome ex- roundworms (nematodes) [32]. Additional evidence for long-term tends far beyond the immune system, affecting metabolism and associations between helminths and vertebrates comes from potentially the development of other parts of the biome, including phylogenetic analyses that have uncovered cases where host and worms, or helminths, that inhabit the gut of all vertebrate species helminth groups show similar patterns of speciation [31,33], [23]. Modern sanitation and water treatment practices, the wide- suggesting that helminths and hosts have co-evolved. Hoberg spread use of antibiotics, modern practices in obstetrics (e.g., deliv- et al., for example, considering the coevolutionary history of ery by caesarian section and the use of antiseptics), and the use of tapeworms and vertebrates, estimated that these helminths have substitutes for mother’s milk, have all profoundly affected the co-evolved with their vertebrate hosts for a minimum of 350– microbiome. These changes almost certainly contribute to hyper- 420 million years [34]. Additional assessments of helminth–verte- immune-associated disease [24,25], but it is profound changes in brate gut associations by molecular clocks or by the geographic colonization of the gut by helminths rather than changes in coloni- distribution of living species relative to tectonic events indicates zation of the gut by microorganisms that has been identified as the that helminth–vertebrate associations are at least 100–200 mil- single most important factor impacting epidemics of hyperim- lion years old [32,35,36]. mune-associated disease [26–29]. Helminths interact in a complex manner with the immune system of the host as well as with the ‘‘Typical’’ association of helminths with humans microorganisms of the human biome, forming a key component of the highly interdependent network normally associated with An understanding of ‘‘typical’’ helminth colonization in humans the human biome (Fig. 1). This key component of the biome has living in a Neolithic culture might provide some insight into the largely been eradicated in post-industrial society, and a wide range environment for which our immune systems are evolved to func- of evidence suggests that this depletion leaves the remaining com- tion. For several reasons, however, defining such colonization re- ponents of the biome in an unbalanced, relatively unstable, and mains problematic. First, epidemiological data on helminth disease-prone state. associations is often geographically patchy and gathered using 496 S.D. Bilbo et al. / Medical Hypotheses 77 (2011) 494–504 different methods, making generalization difficult. Second, most of and therefore may underestimate true colonization rates [49,50]. the data in humans are not derived from populations living in a Thus, studies evaluating colonization of non-human mammals by Neolithic culture, but rather are based on populations living in helminths suggests strongly that colonization by helminths is the towns and cities, where sanitation and anti-helminthics alter the norm for mammals, despite the fact that humans living in post- incidence of colonization and may affect which species are present. industrial societies have come to regard the absence of resident Third, colonization status differs enormously among human popu- helminths as ‘‘normal’’. lations. Climate and socioeconomic status are two of the largest Well-studied ecological biomes provide parallels that might determinants of these differences, but other factors can have a help understand the dynamics of the human biome and the effects strong influence [37,38]. Because many helminths have complex of biome depletion. Ecological biomes such as grasslands and trop- life cycles involving intermediate hosts such as snails or insects, ical forests reach new, unstable equilibria as a consequence of ra- the local ecology of these other species has a major impact. Activ- pid and prolonged loss of co-evolved components [51]. These ities such as farming and hunting increase the likelihood of contact biomes often contain ‘‘keystone species’’ that, relative to their bio- with vectors of early life history stages of some helminths, and can mass or density, have a disproportionately heavy impact on the also influence colonization rates and species composition. As a ecosystem when lost [52]. We propose that the human biome fol- consequence, the epidemiological data we have are likely repre- lows the same properties and has been rendered unstable by the sentative primarily of the past tens to hundreds of years of human recent loss of a critical, co-evolved component: helminths. This history; they are almost certainly not characteristic of the vast instability is apparent in the widespread, aberrant function of the sweep of time humans have been living in association with immune system in the absence of ‘typical’ helminth colonization, helminths. and suggests that helminths might collectively be considered as Despite these uncertainties, it seems clear that colonization by the equivalent of a keystone species of the human biome. multiple helminth species would have been nearly universal throughout human evolutionary history [39]. The most ancient di- Modulation of the immune system by helminths rect evidence comes from two frozen humans dating to the fifth century BCE that contain embryos of hookworms [40]. However, Helminths have evolved to secrete dozens if not hundreds of physical evidence of this kind is rare, and the most extensive evi- molecules that down regulate their host’s immune system dence comes from epidemiological studies. Over 340 species of [53,54]. The range of immunoregulatory molecules includes a vari- helminths colonizing humans have been recorded [41], with about ety of protease inhibitors, including metalloprotease inhibitors, 30 of those species regarded as medically relevant. Rates of coloni- toll-like receptor signaling inhibitors, a variety of antioxidant en- zation are very high for some helminth species even today [41,42]. zymes, cytokine homologs, and a wide range of other molecules For example, more than one-sixth of the world’s population is that inhibit or regulate the function of T-cells, macrophages, and inhabited by just two soil-transmitted helminths, Ascaris lumbrico- dendritic cells [53]. Each species of helminth secretes different pro- ides and Trichuris trichiura, and several other helminths each inha- teins that often exhibit strong host specificity, and many helminths bit more than 100 million humans [41,43]. In some parts of the secrete different proteins at different stages of their life cycle. Hel- world, colonization by multiple helminths is the norm rather than minths also produce non-protein molecules such as phosphoryl- the exception [37,42]. Perhaps the most useful information regard- choline, glycans, and lipids which down-regulate the immune ing normal colonization status comes from indigenous popula- system. It seems inevitable that the host has adapted to the pres- tions, where the influence of confounding factors such as ence of these immunosuppressive ‘‘drugs’’ [28]. sanitation, anti-helminthics, and exotic food sources are minimal. Immunosuppression of the immune system by helminth-de- For example, a study of people living in a pre-industrial culture rived factors is only one means by which helminths might avert in Rwanda revealed serological evidence of helminth colonization allergic and autoimmune reactions. Another very important prop- (average IgE levels in serum above normal for post-industrial soci- erty of helminth colonization that seems almost at odds with the ety, despite the absence of allergy), even in those individuals which immunosuppressive effect is that it causes a substantial and did not test positive for helminths based on a fecal analysis [44,45]. chronic immune response, which probably has several effects on Collectively, the epidemiological evidence strongly suggests that the host. For example, by inducing chronic commitment of the im- humans have been living with multiple helminths throughout their mune system toward helminth-derived factors, helminths leave evolutionary history, and that these associations are a normal part the immune system with fewer resources available for allergic or of our ecology. autoimmune responses. Further, chronic stimulation of the im- Although the typical colonization of humans by helminths mune system by helminths results in the induction of a substantial might be difficult to access with certainty, the number and diver- feedback inhibition system [55,56]. In laboratory rodents, this is sity of helminth species has been assessed in a variety of wild readily observed as an increased production of T-regulatory cells (i.e., not laboratory bred or domesticated) animals, and may pro- following colonization by helminths [53,57]. With these observa- vide insight into what is typical of a mammalian species. Most tions in mind, it seems evident that the ubiquitous presence of hel- individual animals in natural environments are colonized by both minths and their omnipresence during the evolution of the a wide diversity and a larger number and intensity of helminth immune system have profoundly shaped the biology of both hel- species. Although geographic and ecological variations exist in minth and immune system [58], although many of the effects of the patterns of colonization, what is clear is that helminth coloni- this coevolution probably remain to be discovered. zation is the norm. From a broad dataset consisting of 56 mammalian species, in any given population, the number of helminth Biome depletion as the cause of epidemics of hyperimmune- species was found to range from 2 to 39 species [46,47]. Further, associated disease, but not the sole cause of hyperimmune- rates of colonization by helminths are typically high in mammalian associated disease species closely related to humans, including the great apes [48]. For example, it is clear that our closest living relatives, common Almost paradoxically, The Depleted Biome Theory does not im- chimpanzees and bonobos, are both host to a diverse set of hel- ply that biome depletion is the primary cause of allergies or auto- minths in their natural habitats [31,48]. Each helminth species immune diseases. In addition to biome depletion, two other may be present in up to one-third of the individuals in these great equally important factors are often or perhaps even usually found ape populations, although sampling is exclusively based on feces to contribute to hyperimmune-associated diseases (Fig. 2). First, S.D. Bilbo et al. / Medical Hypotheses 77 (2011) 494–504 497 hyperimmune-associated diseases are often associated with a de- a variety of allergic diseases, are explained well by The Biome gree of heritability, having genetic and possibly epigenetic factors Depletion Theory, despite the presence of environmental triggers that predispose people to disease. Second, hyperimmune-associ- and genetic and/or epigenetic factors associated with the disease. ated diseases are often triggered by environmental factors that in- Indeed, it is counterintuitive to attribute recent epidemics of dis- clude viral infections, chemical pollutants, or common antigens ease solely to factors that have remained relatively unchanged produced by a wide range of organisms, ranging from ragweed to for hundreds or even thousands of years, such as human genetics, cockroaches to cows and dust mites. Thus, the Biome Depletion ragweed pollen, wheat proteins, and common viruses. Rather, Theory involves a ‘‘three-hit paradigm’’ that is gaining increasing acute increases in disease must be attributed to factors such as support. Hay fever, for example, is associated with genetic factors biome depletion that are very recent occurrences. Unfortunately, [59], and is, of course, triggered by exposure to antigens such as failure to resolve this issue has resulted in considerable confusion those produced by ragweed. Further, chemical pollutants probably in the biomedical literature: The identification of neither genetic affect the incidence and the severity of hay fever [60]. Thus, biome factors that predispose to disease nor environmental factors that depletion by itself does not actually cause hay fever. However, hay trigger disease rules out The Biome Depletion Theory as applicable fever is a disease only associated with post-industrial society [61], (Fig. 2). Rather, it is (a) the role of ‘‘overly aggressive’’ immune and ongoing studies indicate that it is biome depletion which posi- reactivity, and (b) the increased incidence of disease in post-indus- tions the immune system to react toward harmless (non-patho- trial society relative to pre-industrial agrarian or hunter gatherer genic) antigens [62,63]. Thus, the epidemic of hay fever, like societies that are the hallmarks of diseases associated with biome other hyperimmune-associated diseases, is apparently caused by depletion. biome depletion, even though biome depletion is not sufficient by itself to cause hay fever. Hygiene and the roots of The Biome Depletion Theory Multiple sclerosis is an example of a hyperimmune-associated disease that can be triggered by viral infection [64], is associated The Biome Depletion theory evolved from an idea first devel- with certain genetic markers [65], but yet can be effectively treated oped in the 1980s that has long been known as ‘‘the hygiene by colonization with helminths [55,56]. Similarly, type 1 diabetes hypothesis’’. This hypothesis was based on the understanding that is potentially associated with specific proteins found in the diet certain hyperimmune diseases, allergies in particular, were associ- [66,67] and is associated with certain genetic factors [68]. Yet, hel- ated with components of modern culture that generally increase minth colonization is an effective treatment for type 1 diabetes in hygiene, such as the use of modern, flushing toilets. As the under- autoimmune prone mice [69], and the increase in prevalence of standing of hyperimmune-associated diseases evolved, it was type 1 diabetes in post-industrial cultures has not been explained eventually recognized that epidemics of allergies share a common by the introduction of new proteins into the diet or by a change in cause with epidemics of autoimmune diseases. More importantly the genetics of the population. Thus, epidemics of multiple sclero- for our understanding of the nature of hyperimmune-associated sis and type 1 diabetes, as well as other autoimmune diseases and diseases, changes in the microbiome and, more importantly,

Fig. 2. ‘‘The Biome Depletion Theory’’, with a three-hit paradigm of hyperimmune-associated disease. In this model, three factors interact to induce pathogenic allergic and autoimmune responses. While hygiene generally reduces the presence of one factor, ‘‘triggers’’, hygiene is the source of another factor, biome depletion and subsequent immune hypersensitivity. Of importance are the ideas that (a) ancient factors (triggers, genetic and epigenetics) interact with relatively recent biome depletion to cause epidemics of hyperimmune-associated disease, and (b) the effects of epigenetic factors on disease might not be evident until one or possibly more generations after biome depletion first occurs. 498 S.D. Bilbo et al. / Medical Hypotheses 77 (2011) 494–504 depletion of helminths from the human biome were identified Treatment of epidemics of allergy and autoimmune disease as the leading suspects affecting epidemics of these diseases with biome reconstitution: results so far [29,70,71]. This final revelation clarified the particular component of hygiene responsible for epidemics of hyperimmune-associated It stands to reason that if biome depletion has caused epidemics disease. It was, in fact, not hygiene, per se, that was responsible. In- of allergies and autoimmune diseases, then restoration of the deed, increased exposures to a variety of components associated biome should result in an end of those epidemics. Consistent with with an unhygienic environment, including viral infections, mold, this reasoning, a number of experiments using animal models indi- dust mite-derived allergens, and cockroach-derived allergens, are cate that biome reconstitution does indeed prevent and even in known to be ‘‘triggers’’ for the induction of allergy and/or autoim- some cases treat hyperimmune-associated disease [26–29]. For mune disease. Thus, a particular house might be considered unhy- example, experimentally induced colitis, experimentally induced gienic in some regards because of high concentrations of mold or allergy, and type 1 diabetes can be effectively averted or treated cockroach-derived allergens, which increase the propensity for in rodents by colonization with Heligmosomoides polygyrus,a allergic disease. At the same time, in the same house, the environ- roundworm commonly found in the small intestine of a variety ment might be considered relatively hygienic because a modern, of rodents [69,74,75]. Experimentally induced allergy in rodents flushing toilet and running water are used regularly, which, as dis- can also be alleviated by colonization with another roundworm cussed below, effectively disrupts the life cycle of almost all hel- Nippostrongylus brasiliensis [76]. minths, further increasing the propensity for allergic disease. Even more encouraging than studies using animal models are Therefore, hygiene per se does not have a predictable effect on two particular studies in humans. First, clinical trials demonstrate hyperimmune-associated disease: Hygiene in the form of a toilet that exposure to a porcine helminth, Trichuris suis, provides an increases the risk for hyperimmune-associated disease, while effective treatment for many patients with inflammatory bowel hygiene in the form of preventing buildup of mold and cockroach disease whose disease had proven untreatable with modern med- derived allergens reduces the risk of hyperimmune-associated icine [77]. More exciting results show that the progression of mul- disease. Failure to understand this distinction has unfortunately tiple sclerosis is halted by accidental helminth colonization [55]. retarded progress in the field. A number of clinical trials addressing a variety of hyper-immune disorders are currently underway utilizing the porcine whip- The timing of biome depletion and hyperimmune-associated worm, T. suis, which has proven useful in treating inflammatory epidemics bowel disease, as mentioned above. Importantly, this helminth is adapted to colonize pigs, and does not colonize humans. Rather, The use of toilets effectively blocks the life cycle of almost all it persists in the human intestine for a few days to weeks before helminths, rendering humans in post-industrial societies as the being eliminated, and must be re-introduced to the human intes- only vertebrates free of association with these organisms to a tine on a frequent basis to maintain therapeutic levels. It is poten- large extent. The few helminths with the ability to reproduce tially difficult to place these pioneering clinical trials with T. suis despite the use of toilets, such as pinworms, are easily defeated within the broader perspective of biome reconstitution. It remains by modern medicine because their ability to evolve resistance unknown, for example, how exactly exposure of a patient to T. suis to pharmaceuticals is relatively limited by their long life cycle might affect any subsequent attempts to reconstitute the biome of and small population size in comparison to bacteria and viruses that patient with helminths that are adapted to colonize the hu- that readily evolve drug resistance. Despite the fact that many man intestine. It also remains unknown how effective T. suis is in individuals in post-industrial societies today take for granted comparison to a variety of other helminths that are adapted to hu- the presence of toilets, the introduction of that technology is very mans, and if T. suis might work in combination with other hel- recent, occurring, for example, in urban environments less than minths as a therapy. Further, given the high cost of T. suis, due in 100 years ago in the United States, and much more recently in large part to the need for frequent re-exposure to the organisms, rural environments. Most importantly, the impact of biome- it seems an unlikely candidate for use in clinical trials aimed at depleting technologies such as the toilet on the immune system prophylactic reconstitution of the biome, if such trials are under- is expected to be delayed rather than immediate. This delay taken. Regardless of the utility or lack thereof that T. suis eventually between cause and effect is anticipated for several reasons. First, affords modern medicine, work with T. suis represents the very first many helminths have a long life span, some living for decades, pioneering efforts in what promises to be a large and productive and thus many individuals are expected to remain colonized for field of clinical medicine. extended periods even after the transmission of the helminth has been halted. Second, some aspects of immunity may be The health costs of biome depletion: how much? strongly influenced by colonization with helminths early in life, and these influences may not be entirely reversed by loss of Some might argue that treating hyperimmune-associated dis- colonization at a later time. Thirdly, an individual’s immune eases such as hay fever and even food allergies is not worth the system is strongly influenced by their mother’s immune system, time, effort and energy that will be required to intentionally and and the extent to which the biome of the mother might protect systematically re-introduce organisms classified as ‘‘parasites’’ into the offspring from hyperimmune-associated disease remains the human population. Indeed, it can be argued that hay fever may unknown. The profound impact of epigenetics on human biology be a worthwhile price to pay for avoiding these coevolutionary is only now beginning to be understood [72,73], and, although partners. Crohn’s disease and multiple sclerosis are certainly less speculative, epigenetic effects resulting from colonization with tolerable, but therapies which usually alleviate the symptoms of helminths might be seen for multiple generations, thus delaying these diseases at least to some extent are available. Despite the at least some of the effects of biome depletion (Fig. 2). With these generally high costs of many pharmaceuticals, the general consen- factors in mind, it is not surprising that the effects of biome sus may be that some disease can be tolerated as a side effect of depletion are apparently increasing at the present time, despite being parasite free. However, the apparently higher cost of biome the fact that for many, it was their grandparents, not themselves, depletion undermines this argument. which first enjoyed the convenience of biome-depleting technol- Although hyperimmune-associated diseases are not perceived ogies. In fact, we do not yet know if the potential effects of biome as particularly life threatening, the death toll is not trivial. Allergic depletion have reached a climax. reactions to food alone kill an estimated 150–200 people every S.D. Bilbo et al. / Medical Hypotheses 77 (2011) 494–504 499 year [78] Added to this number are the more than 3000 people immune cells of the brain [95], and increased expression of pro- who die from asthma each year [14,79]. In addition, if the current inflammatory cytokines (e.g., interleukin [IL]-1b; [96]) which in epidemic is not abated, more than one of every 300 people will turn may influence neural function (e.g., long-term-potentiation have their lives strongly impacted if not shortened by lupus, multi- (LTP); [97]). Cognitive decline is one of the primary consequences ple sclerosis, or type 1 diabetes. The number of deaths associated of normal aging, although there is significant individual variability, with colitis and other types of gastrointestinal inflammatory dis- as some individuals age more successfully than others, with a sub- eases adds to this number. set of the population eventually developing dementias such as Alz- Another factor when considering the health costs of biome heimer’s disease [98,99]. Again, the presence of depletion is that we do not yet know the extent of pathology that neuroinflammatory changes in normal aging suggests the possibil- may result. Diseases not obviously related to a hyperimmune re- ity that hyper-immune activity leading to excessive neuroinflam- sponse may be impacted by biome depletion. For example, biome matory mechanisms can initiate poor cognitive aging. The reconstitution in mice attenuates helicobacter-induced gastric potential influence of biome depletion on age-associated dementia atrophy [80], suggesting that biome depletion may increase the remains unknown, but investigation of the possibility seems propensity for gastric atrophy, which is a precursor to stomach warranted. cancer, the second leading cause of cancer-related deaths in hu- Based on the above considerations, we suggest that biome mans. The overall analysis leaves us uncertain of the price we are depletion is changing the immune system at a population-wide le- currently paying for biome depletion, and in an uneasy ‘‘wait- vel, which will undoubtedly impact the brain, particularly during and-see’’ position, not knowing what immune-associated epidem- sensitive periods of development and perhaps in the aging process. ics are yet to come. This uneasy position is made worse by the real- Importantly, this theory does not compete with any prior hypoth- ization that the effects of biome depletion may strongly impact eses or known risk factors for neuroinflammatory conditions, but human brain function. rather complements them. For instance, biome depletion may set the stage for exaggerated levels of neuroinflammation, which Biome depletion and cognition interacts with other known risk factors (stress, infection, or genetics) in the induction of a given disease or disorder. Our brain is intertwined with our immune system in ways that we are only beginning to understand [1,81]. Immunocompetent The physiologic effects of biome depletion cells are located throughout every organ of the body, including the brain, and sophisticated interactions occur among these cells, Although some of the consequences of biome depletion for primarily via soluble protein messengers called cytokines [82,83]. health are becoming apparent, the exact nature of the effects of Notably, bidirectional communication between the brain and im- biome depletion on the immune system remains unknown, even mune system by cytokines has significant consequences for neural in laboratory rodents. Virtually all research on the immune system function and behavior, including social, cognitive, and affective has been conducted in biome-depleted (helminth free) laboratory abilities. For instance, sick animals exhibit several well-character- animals or in humans living in a post-industrial society. Because ized behavioral changes, including reductions in activity, explora- of this, immunologists are now faced with the unsettling realiza- tion, and social and sexual interactions, as well as cognitive and tion that the immune system they have spent all of their effort affective changes [84,85]. Despite their maladaptive appearance, and energy dissecting over in the past 50 years is dramatically dif- these behaviors are organized, adaptive strategies that are critical ferent than any immune system in a natural state of existence. In to host survival [86,87]. fact, ‘‘normal’’ is not helminth-free, and our coevolutionary part- However, increasing evidence has linked chronic, exaggerated ners must be included if we want to understand and appreciate cytokine expression and resulting neuroinflammation within the the ‘‘normal’’ state of immunity. brain to pathologic conditions as diverse as Alzheimer’s disease, Several factors potentially complicate efforts to determine the autism, and depression—notably, all conditions that share some physiologic consequences of biome depletion. First, not only are striking similarities to sickness behaviors that have somehow be- the effects of biome depletion on a particular generation undoubt- come prolonged or pathological [88]. Neuroinflammation, for edly profound, the effects of biome depletion on subsequent gener- example, is strongly suspected as having a causal role in autism ations, passed down by epigenetic mechanisms, might be as well as a number of other developmental disorders. Further, considerable. The fact that the immune system of the fetus is neuroinflammation may be extremely important during the peri- strongly influenced by the immune system of the mother particu- natal period, potentially altering the ‘‘program’’ of the normal larly supports this idea. In addition, the transfer of immune compo- course of development, with the result that adult outcomes, nents through the mother’s milk adds to the potential generational including behavior, are significantly and often permanently altered effects of biome depletion. A second factor which complicates ef- [89]. This idea accounts for the observation that perinatal exposure forts to determine the physiologic consequences of biome deple- to infectious or other immune activating agents (e.g., stress, envi- tion is the fact that biome depletion is not an all-or-nothing ronmental toxins) have a number of enduring influences on phys- proposition. The idea that specific components of the biome have iology and behavior, including reactivity to stress, disease different niches exponentially increases the complexity of efforts susceptibility, and notably, increased vulnerability to mental ill- to evaluate the effects of biome depletion. The fact that those indi- ness such as autism and schizophrenia [90–94]. Thus, to the extent vidual components of the biome interact with other components of that biome depletion results in an increased proinflammatory the biome as well as with the host [100] adds even more complex- environment during the perinatal period, biome depletion is ex- ity to the issue. pected to cause an increase in the prevalence of mental illness. A wide range of studies in laboratory animals indicate that hel- In support of this idea, a variety of indicators, including epidemio- minths exert a potent effect on virtually all components of the im- logical, molecular, and genetic aspects of disease, suggest that mune system [53]. Studies from our laboratory using a wild-caught biome depletion is responsible for the increasing incidence of aut- rat versus laboratory rat model have provided insight into the po- ism [7]. tential extent of the impact of biome depletion on the immune sys- A large number of neuroinflammatory changes have also been tem [45,101]. The differences between the immune systems of identified in normal brain aging, including upregulation of major wild rats and those of laboratory rats exceed many inter-species histocompatibility (MHC II) expression on microglia, the resident differences in immunity. For example, wild rat derived lymphocytes 500 S.D. Bilbo et al. / Medical Hypotheses 77 (2011) 494–504 demonstrate an unresponsiveness that is not found in lymphocytes immunoregulatory molecules by living helminths may be impor- derived from laboratory rats, laboratory mice, or humans in a post- tant for their function, and it is possible that new drug delivery industrial society [101]. Similarly, studies comparing the immune technologies may need to be devised which mimic the delivery systems of wild mice with laboratory mice [102] also show a wide of a living helminth. In other words, given the long coevolution range of differences in baseline immune parameters and immune of immune systems and helminths, it seems reasonable that the responsiveness. To the extent that the biome is, in fact, the primary immune system may be adapted to and indeed dependent upon driving force behind the difference between ‘‘biome-normal’’ wild the nature, dose, and delivery method used by living helminths. rodents and biome-depleted laboratory rodents, ongoing results Probably it is the field of evolutionary biology that most indicate that biome depletion throws a wide range of parameters strongly argues for living helminths and biome reconstitution in associated with cellular and humoral responses into disarray. For favor of pharmaceutical substitutes. Helminths represent a poten- example, the overall maturation of the immune system as well tial therapeutic that has been fine-tuned by hundreds of millions of as the balance between innate and adaptive immunity may be years of natural selection not to encumber a healthy host. Consis- profoundly altered by biome depletion. As pointed out recently by tent with this idea, several helminths are known to cause few if Maizels and co-workers [53], helminths can regulate ‘‘every aspect any symptoms in most cases [55,107,108]. The bovine tapeworm of host immunity from initial recognition to end-stage effector (Taenia saginata), for example, has been labeled a ‘‘commensal’’ mechanisms’’. Traditional paradigms associated with allergy and rather than a parasite in humans [107]. To achieve this balance, autoimmune disease (e.g., Treg versus Th1 versus Th2 versus natural selection has tested countless billions of combinations of Th17) do not adequately address this effect. molecular tools over millions of years, selecting those that are most effective for both helminth and host survival. Quite obvi- To reconstitute the biome or to develop helminth-inspired ously, no pharmaceutical has ever been developed to match that drugs that mimic a reconstituted biome? record. Another advantage that living helminths have over pharmaceu- Biomedical researchers often envision isolation and character- ticals is their potential cost effectiveness. Helminths are typically ization of the individual components produced by helminths that long-term therapeutics if used in their natural host species, with modulate the immune system, with the goal of creating new hel- a single dose lasting for years if not decades. Thus, costs will be minth-inspired drugs to treat disease. On the one hand, this type limited by the long-term effectiveness of a single treatment. Fur- of approach with other organisms has proven instrumental in the ther, the prospects for new technologies that facilitate cost effec- progress of modern medicine, giving us antibiotics and some tive and safe production of helminths in sufficient numbers for important vaccines. Further, repeated injection of helminth-de- widespread use seem almost unlimited. Biotechnology involving rived extracts or proteins in animal models of disease has already in vitro culture of helminths or cultivation of human specific hel- proven useful in alleviating such conditions as collagen-induced minths in genetically modified mice (e.g., humanized or immuno- arthritis [103] and experimentally induced colitis [104], among suppressed mice) are two examples of such biotechnologies which others [105,106]. On the other hand, several factors are likely to remain untapped. The technology and clinical implementation of limit the general use of this approach as a substitute for biome biome reconstitution may eventually prove no more complex than reconstitution. that utilized for vaccinations, with biome reconstitution and main- First, it is difficult to imagine a single pharmaceutical or even a tenance being a routine part of a typical checkup at the doctor’s collection of pharmaceuticals that could recapitulate the vast com- office. plexity of the interaction between helminths and the host immune One potential advantage of living helminths as a therapeutic is system. While pharmaceuticals are generally directed at one com- that they might be genetically altered to enhance their beneficial ponent in the immune apparatus, a single helminth species pro- nature. Currently available organisms can hypothetically be modi- duces dozens if not more molecules that each target specific fied by selective breeding to enhance their beneficial nature, or components of host immunity. The likely possibility that more may be modified in the laboratory to create specifically altered than one species may be required to effectively reconstitute the organisms, including transgenic helminths. In this manner, genet- biome adds a level of complexity that seems staggering to anyone ically modified helminths could be used as an effective drug deliv- interested in replicating the natural state using pharmaceuticals. ery agent (vector) to treat a potentially unlimited variety of Perhaps more importantly, our understanding of helminth immu- medical conditions. Helminths offer a number of advantages over noregulation is far from complete, with many gaps in our knowl- viral or bacterial vectors for certain applications because of their edge [53]. For example, many proteins secreted by helminths in long life span, built in resistance to destruction by the immune sys- low abundance may not have been identified, but yet may have tem, and, since they do not complete their life cycle in the host, a important biological activity. Further, the biological role of some low propensity to mutate or evolve once introduced into the host. proteins secreted by helminths in high abundance, such as the gly- One potential objection to the use of mutualistic helminths for colytic enzyme triose phosphate isomerase, remains poorly under- biome reconstitution is that the introduction of organisms possess- stood [53]. Thus, our present level of understanding of the vastly ing ‘‘vastly complex and uncharacterized elements’’ into the hu- complex interactions between helminths and their hosts poten- man body might have unforeseen consequences. This argument tially adds a substantial hurdle to the design of therapeutics which is easily turned on its head, however, when we ask how it is ra- will mimic those interactions. tional to eliminate our coevolutionary partners that possess vastly Not only is the helminth/host interface vastly complex, it re- complex and uncharacterized elements without expecting dire quires continuous input from the helminth. Helminths actively consequences. Indeed, many of the factors proven to be important maintain a state of immunosuppression, and removal of helminths for human survival, including the microbiome and even the food results in a loss of that regulation [53], suggesting that any phar- we eat, contain vastly complex and uncharacterized elements. maceutical substitute would also need to be maintained at a rela- Thus, complexity and a lack of understanding of that complexity tively constant level for maximum efficacy. Such continuous do not rule out the importance of an element of the normal human maintenance is potentially difficult and costly to maintain using biome for health. Further, many pharmaceuticals, molecules with traditional pharmaceuticals, and regular injections of helminth-in- substantially less complexity than helminths, cause exceedingly spired drugs, even if effective, might prove burdensome and complex reactions within the human body, often with consequences expensive. Further, the mechanism of delivery of helminth-derived that remain poorly understood. Thus, the relative simplicity of a S.D. Bilbo et al. / Medical Hypotheses 77 (2011) 494–504 501 drug at the molecular level does not necessarily lead to a complete diet and little exercise. However, just as post-industrial societies understanding of that drug’s effects, and certainly does not impart do not need to face predation and possible starvation to avoid safety for patients. Finally, the idea of biome reconstitution is not obesity and heart disease; neither do post-industrial cultures need to modify human biology in a new way, but to restore the ecology to face the specter of helminth-associated disease in order to avoid of the post-industrial human biome to harmony with the way that allergy and autoimmune disease. Just as we have the option of our genes have evolved to function. In a nutshell, comfort with the safely utilizing proper diet and exercise regimens to give our car- idea of biome reconstitution requires only an understanding that diovascular system what it has evolved to require, it is expected natural selection has shaped our genes so that they are compatible that, far more effortlessly, we can safely utilize selected organisms with the environment. It does not require a complete and total to give our immune system what it has evolved to require. understanding of that environment or how that environment interacts with the human body. Biome reconstitution and the public consciousness Another potential objection to the concept of biome reconstitution is the fact that helminths extract a horrible toll of suffering A commonly expressed concern regarding biome reconstitution and death in developing countries [109]. Given this potential to regards the ability of those who would benefit from the therapy to cause harm, it could be argued that these organisms should not actually accept the therapy. Some speculate that the prospect of be reintroduced into post-industrial cultures. This argument, how- helminth colonization will prove too repulsive for most patients ever, is entirely undermined by a consideration of the nature of to accept. However, from several perspectives, it can be argued that parasitic disease in developing countries. In developing countries, the view of the public toward the concept of biome reconstitution at least one of the following three conditions are necessary ingre- will pose no obstacle whatsoever. Most importantly, it is expected dients for morbidity and mortality associated with helminth that biome reconstitution will be the subject of extensive clinical infection: trials, and will have a proven safety record prior to widespread use. In the face of clinical studies potentially showing that biome 1. The widespread occurrence of starvation and malnourishment, depletion can lead to lupus, multiple sclerosis, food allergies, asth- conditions which add risk to colonization by helminths. ma, and perhaps even an autistic child, it seems far more likely that 2. The absence of water treatment facilities and sewer systems, concern over biome depletion rather than fear of biome reconstitu- allowing uncontrolled infections and potentially high burdens tion will be the hallmark of public opinion in the future. It seems of helminths. inconceivable that an educated public would react with paranoia 3. The presence of helminths that are not well adapted to the host toward helminths rather than with concern for their health and (e.g., parasites that are harmful to even a healthy host, such as the health of their children. Certainly the educated public has not Dracunculus medinensis, which causes dracunculiasis, Taenia reacted with paranoia toward probiotics. Indeed, the public is solium, which causes cysticercosis, and Loa loa, which causes becoming aware of the dangers to their microbiome imposed by Loa loa filariasis). the heavy use of antibiotics. Further, no responsible individual plans to market helminths as a can of worms any more than probi- Not one of these factors presents a difficult hurdle when consid- otics or vaccinations have been marketed as a can of germs. There ering the reintroduction of helminths into post-industrial societies, seems to be little evidence to support the idea that revulsion of which generally enjoy over-nourishment rather than malnourish- helminths will be an impediment to biome reconstitution. ment. Given a proper selection of helminths for colonization, Reassuringly, negative views toward helminths that might be uncontrolled infections are impossible in the face of modern sewer adopted by a few individuals are not expected to influence the effi- systems and water treatment facilities. Further, modern medicine cacy of biome reconstitution for the population as a whole. As with makes it possible to easily identify individuals with pre-existing diet and exercise programs, the health benefits of biome reconsti- conditions that could be contraindications for helminth therapy tution will be limited to those who embrace the prospect, and one (e.g., anemia, malnutrition, immune deficiencies, coagulopathies, patient’s health will not be influenced by another’s decision to em- advanced aging, etc.). In addition, modern clinical research prac- brace or reject biome reconstitution. Fortunately, biome reconsti- tices can readily be utilized to ensure that adverse side effects tution should eventually prove much easier for the average are reduced to far below acceptable levels prior to widespread individual to implement than does an effective diet and exercise therapeutic application. Thus, post-industrial cultures have the program, since biome reconstitution is potentially a matter easily luxury of selecting controlled colonization with helminths, not handled by a physician during a routine medical exam. pathologic infection. In essence, post-industrial society will have domesticated helminths. No restoration of the natural ecology of helminths is expected or even suggested, with the survival of the Approaching the solution domesticated helminths being strictly dependent on the medical community. To maintain a reconstituted biome, patients would The approach to biome reconstitution needs to be systematic need periodic evaluation and maintenance, since natural transmis- and rapidly executed. The following questions need to be ad- sion of therapeutic helminths would be non-existent given the dressed with the utmost urgency: proper selection of helminths and the sewer systems of post- industrial culture. Further, given the effectiveness of anti-helmin- 1. Which helminths or combination of helminths are safe and thic drugs, therapy will be readily reversible should particular pa- effective? Several species, including the bovine tapeworm (T. tients develop a condition in which the helminths are no longer saginata), the human whipworm (T. trichiura), and the human warranted. hookworm (Necator americanus) are well adapted to humans, The loss of evolutionary partners leading to disease is not un- asymptomatic in low numbers, and thus seem to be obvious ique to the helminth/hyper-immune condition. The historically candidates for initial consideration. New technologies might omnipresent struggle against predators and for food resources be developed (e.g., irradiation of organisms to achieve sterility have left our bodies addicted to particular diets and a degree of and eliminate the possibility of transmission, new culture exercise. The loss of predators and the loss of any challenge in find- methods, or cultivation of human-specific helminths in ultra- ing food in post-industrial cultures have led to epidemics of obes- clean and immunodeficient rodents) which could improve on ity, type 2 diabetes, and other complications associated with a rich the initial answers to this question. 502 S.D. Bilbo et al. / Medical Hypotheses 77 (2011) 494–504

2. Which helminths should be utilized, at what dose, and under basic immunology decide to take action, biomedical research can what conditions (patient age, disease state, gender, etc.)? Biome move with astonishing speed. reconstitution for a 3-year old patient may be quite different After that tipping point is reached for biomedical research, that that required for a 30-year old patient, and the best financial considerations could drive a very rapid evolution of clin- approach to biome reconstitution for children may depend on ical medicine. For example, if restoring the biome of a few hundred the status of the mother’s biome during pregnancy and the first patients is shown to prevent the onset of even a few cases of such years of the child’s life. costly diseases as asthma, type 1 diabetes, or autism, strong pres- 3. Which hyperimmune-associated diseases can be cured or effec- sures on financial resources could force the health care system to tively treated with biome reconstitution, versus which can only adapt quickly. With that in mind, the day when a doctor will pre- be prevented by biome reconstitution? The answer to this ques- scribe not only a vaccination against a dreaded disease, but also tion should drive efforts (or the lack thereof) to prophylactically colonization with a domesticated helminth, may not be so far dis- restore the biome in the general population. tant in the future. The best news is that this type of treatment is 4. How important for children is the biome of the mother prior to expected to be both inexpensive and without major complications. and during pregnancy, and while breastfeeding? If we keep open minds about the possibilities of biome reconstitu- 5. What medical conditions (e.g., suppressed immune system, tion – the ‘‘tipping point’’ may be just around the corner. anemia, and coagulopathy) would be contraindications for biome reconstitution? Conclusions 6. Are particular human genotypes better suited to particular helminth loads? Certainly hyperimmune-associated disease is con- It was only a very few generations ago, at most, that humans first nected strongly with genotype, and it is thus reasonable to began widespread use of indoor flushing toilets and acquired access expect that an optimized approach to biome reconstitution to modern medicine. These technologies, which effectively interrupt may need to consider the genotype of the patient. most helminth life cycles, have, over time, resulted in a profound 7. Does modern medicine need to work toward reconstituting depletion of the human biome. Realizing the intimacy and strength components of the biome other than helminths? For example, of the connection between the biome and the immune system, it is should intentional and controlled exposure to a normal micro- possible to arrive at a remarkably simple conclusion that can ac- flora in a manner that recapitulates natural child birth replace count for a wide range of post-industrial disease: Biome depletion current practices in obstetrics? has left us with an overly reactive immune system, leading to epi- 8. Will certain components of modern medicine (e.g., vaccines and demics of a wide range of allergic and autoimmune diseases. immunosuppressive drugs) need to be modified so that they are A synthesis of epidemiology, modern medicine, evolutionary effective on individuals with a healthy (reconstituted) biome? biology and immunology strongly suggests that biome reconstitu- Modern medicine has been tailored to those with a depleted tion and maintenance should be a major thrust of biomedical re- biome, and may require some adjustment for those with a search in the very near future, and a major component of healthy biome. medicine in the foreseeable future. Helminth-derived pharmaceu- 9. What are the effects of biome reconstitution on human biology ticals might offer an alternative to biome reconstitution, but given in general? For example, how does it affect reproduction, aging, what is already known about the complex and multi-faceted nat- cancer, infectious disease, and cognition? ure of helminth–host interactions, pharmaceuticals which effectively mimic the effects of helminths might prove extremely Obtaining the answers to these questions is the beginning of a difficult and expensive to develop and utilize. Further, the low new chapter in modern medicine and biotechnology, and poten- costs, high efficacy, and very low rate of side effects that are ex- tially closes the door on a chapter aimed at treating hyperimmune pected to be associated with biome reconstitution make this ap- associated diseases with pharmaceuticals. The answer to these proach extremely appealing. In addition, the potential for questions will potentially drive a substantial arm of clinical medi- helminths to be used prophylactically and the potential for new cine for the foreseeable future. Given the current mortality, helminth-supported biotechnology applications add to the attrac- morbidity, and expense inflicted by epidemics of hyperimmune- tiveness of biome reconstitution. The domestication of helminths, associated disease, speed seems paramount. At the same time, as with the domestication of other animals, may eventually pro- however, a systematic rather than a piecemeal approach to the vide a means for selection and breeding of organisms that have a solution is strongly encouraged. greater utility for humans than those helminths found naturally. The biology imposed by our evolution is, at present, inescap- Prognosis for modern medicine able, and medical science of the future will take that fact fully into account. To achieve this aim, intensive and systematic research One question in the minds of many who recognize the vast po- should now be focused on the wide range of questions regarding tential that biome reconstitution appears to offer is how quickly the effects of biome depletion and the application of biome recon- modern medicine will take advantage of this potential. From sev- stitution. Hopefully, in the future, our biomes will be checked as eral perspectives, the prognosis for modern medicine is very good. regularly as our cholesterol is checked, with proactive as well as Modern immunology has proven to be a highly flexible discipline reactive biome reconstitution being routine. as it struggles against widespread epidemics of immune related disease. Within a few short years of initial discovery, new findings Conflict of interest statement often become ‘‘hot’’ topics, accruing well funded laboratories and hundreds of research papers. With that in mind, a tipping point The authors have no financial or other relationship with people in medicine can be seen fast approaching, in which colonization or organizations that may inappropriately influence the author’s with helminths becomes the next hot topic of basic and clinical work. research. The critical events that mark this tipping point are not likely public opinion or even our capacity to solve the Acknowledgements problem, but rather a change of paradigm within the minds of the policy makers of the biomedical community. When those S.E.P. is supported by an European Union FP7 Marie Curie Fel- who strongly influence the direction of the fields of clinical and lowship. The authors acknowledge the contribution of the National S.D. Bilbo et al. / Medical Hypotheses 77 (2011) 494–504 503

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