Supporting Information



Supporting InformationSupplementary Figure SEQ Supplementary_Figure \* ARABIC 1Supplementary Figure SEQ Supplementary_Figure \* ARABIC 1 Calculation of antibody responses for Live Bacterial FACS and ELISA in arbitrary units [AU] according to the standard curve. The same plasma sample of a healthy donor with strong commensal-specific antibody responses was included as a standard in every Live Bacterial FACS assay (A and B) and ELISA (C and D). Serial dilution of the standard plasma (A and C) allowed calculating a standard curve by plotting the plasma concentration in % against the measured median fluorescence intensity (B) or optical density OD405 (D) on a double-log scale. The standard equations (B and D) were calculated with Excel (Microsoft Office) by Trend/Regression Type “Power”. The signal intensity of the undiluted standard plasma was arbitrarily set to “1”. Signal intensities measured in serial dilutions of the test samples were used to calculate the magnitude of antibody responses in reference to the standard plasma via the standard equation. If the antibody response of a given test sample was calculated to be “0.5”, this means that the antibody response in the undiluted test sample is half as strong as in the undiluted standard plasma according to the acquired fluorescence intensities.Supplementary Figure 2Supplementary Figure SEQ Supplementary_Figure \* ARABIC 2 Comparison of antibody responses to commensal bacteria between individuals with blood group A/B/AB and blood group 0. IgA, IgG and IgM responses to E. coli, E. faecalis and S. epidermidis were analyzed in healthy donor samples and grouped according to blood antigen reactivity.Supplementary Figure 3Supplementary Figure 3 Comparison of IgG responses against B. japonicum and E. coli in plasma of healthy donors using either ELISA with whole bacteria or lysate. ELISA with plasma of 8 healthy donors was performed using whole bacteria or bacterial lysate (B. japonicum and E. coli isolate). Optical densities (OD405) values against B. japonicum (white) or E. coli (black) are depicted for different plasma dilutions. Each dot represents 1 healthy donor. Statistical comparisons (only comparisons with t-test p-values <0.05 and corresponding Wilcoxon p-values are listed)ParameterComparisonp-values(t-test)p-values (Wilcoxon)total IgGhealthy donors vs. all HIV+***2x10-13***7x10-8healthy donors vs. HIV+ w/o diarrhea***4x10-7***6x10-8?healthy donors vs. HIV+ with diarrhea***8x10-8***3x10-6total IgMhealthy donors vs. all HIV+***1x10-4***0.001healthy donors vs. HIV+ w/o diarrhea***5x10-4**0.002?healthy donors vs. HIV+ with diarrhea**0.001**0.009E. faecalis IgAIBD vs. healthy donors*0.02**0.002E. coli Nissle IgG?healthy donors vs. HIV+ with diarrhea*0.04***2x10-4S. epidermidis IgGhealthy donors vs. all HIV+***4x10-5***5x10-9healthy donors vs. HIV+ w/o diarrhea***1x10-4***6x10-9?healthy donors vs. HIV+ with diarrhea***2x10-4***1x10-7P. acnes IgGhealthy donors vs. all HIV+***8x10-4***1x10-4healthy donors vs. HIV+ w/o diarrhea**0.001**0.002healthy donors vs. HIV+ with diarrhea**0.002***1x10-4Candida albicans IgGhealthy donors vs. all HIV+*0.04?K.pneumoniae IgGIBD vs. healthy donors***<1x10-4***6x10-4healthy donors vs. HIV+ w/o diarrhea*0.021***6x10-4B.thetaiotaomicron IgGIBD vs. healthy donors*0.020*0.045Supplementary Table SEQ Supplementary_Table \* ARABIC 1usSupplementary Materials and MethodsLive Bacterial FACS Live Bacterial FACS was performed similarly as previously described.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TbGFjazwvQXV0aG9yPjxZZWFyPjIwMDk8L1llYXI+PFJl

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ADDIN EN.CITE.DATA [1] The assay procedure is the same for all bacteria including Candida albicans. All buffers and solutions subsequently used were filtered (22 ?m) to remove contaminating particles which have similar FSC/SSC properties as bacteria. All washing steps included a centrifugation step at 3200 x g for 10 min. Fresh, liquid over-night cultures from bacteria were washed three times with PBS. Bacteria were resuspended in FACS buffer (PBS, 2 % FCS, 5 mM EDTA, 0.05 % sodium acide) to 4x108 bacteria/ml. Bacteria were aliquoted to 1x106 bacteria/well in a 96-well V-bottom plate. Plasma or serum samples were diluted 1:5 to 1:40'000 in FACS buffer depending on the bacteria and 25 ?l were added per well. The FACS buffer used until this step included 1 M Galactose [Sigma] to block natural antibodies with anti-Gal specificity . ADDIN EN.CITE <EndNote><Cite><Author>Galili</Author><Year>1988</Year><RecNum>316</RecNum><DisplayText>[2]</DisplayText><record><rec-number>316</rec-number><foreign-keys><key app="EN" db-id="rexwvprvkax297e2df4pxw9uzxapas909ze5">316</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Galili, U </author><author>Mandrell, R E </author><author>Hamadeh, R M </author><author>Shohet, S B </author><author>Griffiss, J M </author></authors></contributors><titles><title>Interaction between human natural anti-alpha-galactosyl immunoglobulin G and bacteria of the human flora.</title><secondary-title>Infection and Immunity</secondary-title></titles><periodical><full-title>Infection and Immunity</full-title><abbr-1>Infect. Immun.</abbr-1><abbr-2>Infect Immun</abbr-2></periodical><pages>1730-1737</pages><volume>56</volume><number>7</number><section>1730</section><dates><year>1988</year><pub-dates><date>July 1988</date></pub-dates></dates><urls><related-urls><url>;[2] After 30 min incubation at 4°C samples were washed twice with FACS buffer. Incubation with secondary antibodies was done for 30 min at 4°C followed by two washes. The following secondary antibodies were used: goat anti-human IgG (H+L) FITC, goat anti-human IgA (α-chain specific) APC, donkey anti-human IgM Biotin or goat anti-mouse IgG (γ-chain specific) APC [all Jackson Immunoresearch]. Where applicable the incubation step was repeated with Streptavidin-Pacific Blue [Invitrogen]. For flow cytometric analysis samples were resuspended in PBS/2% para-formaldehyde. Data were collected using a LSRII flow cytometer [BD Biosciences]. Data files were analyzed using FlowJo software [Tree Star Inc.]. The magnitude of the antibody responses [AU] was always calculated in reference to the same plasma sample which was included as a standard in every assay. Details are described in Supplementary Figure 1.ELISA107 gentamicin-treated (100?μg/mL) bacteria or 0.1 % bacterial lysate (lysate production with FastBreak? Cell Lysis Reagent, Promega) were coated on ELISA plates (96-well Nunc-Immuno?, Langenselbold, Germany) overnight at 4°C in coating buffer (0.1?M NaHCO3 at pH 9.6). After three washing steps with washing buffer (PBS 0.05 % Tween-20) and blocking with blocking buffer (PBS 10 % FCS) for 2 hours, different dilutions of plasma samples in blocking buffer were added to the wells and incubated for 2 hours at room temperature. Wells were washed four times and thereafter incubated with 100 μl/well HRP-conjugated anti-human IgG-Fc antibody (Bethyl Laboratories) diluted 1:5000 in blocking buffer for 1 hour at room temperature. After washing four times, plates were developed with 100??l/well of a solution containing 0.2?mg/mL 2,2′-azino-di-(3 ethylbenzthiazoline sulfonic acid) (ABTS), 0.1?M NaH2PO4 and 0.04% H2O2 at pH 4. After 10 to 20 minutes plates were read at 405?nm in a Victor3 reader (Wallac 1420, PerkinElmer, Waltham, MA, USA).Statistical AnalysisStability of responses We tested the stability of the individual commensal-bacterial antibody responses in the following way: For each patient, we interpolated the measured data points by stepwise linear functions and calculated the mean of the absolute values of the corresponding slopes. A high value would indicate a large variation in the response. We found that individual bacterial antibody responses only varied between 10-5-10-3 mg/ml/day on average, indicating a quite stable response. To ensure that this rather small change in the bacterial antibody response does not lead to a general in- or decrease of this response over time, we fitted a linear function to the data of a patient measured at multiple time points and tested if the estimated slope coefficient would differ significantly from 0. We predominantly found no indication for a significant in- or decrease of the commensal-bacterial antibody responses over time. (We also tested if the variation in the antibody responses varied between the different study groups and found no generally significant difference). Group comparisons For each member of a study group we calculated the median response of the multiple-time-points measured. Using the median is appropriate as individual commensal-bacterial antibody responses are found to be stable over time. We also performed the analysis with a longitudinal weighted average. This was calculated by the area under the curve, which is formed by a linear approximation of the data, weighted by the time-span for which values were observed. Replacing the median response by the mean or the longitudinal weighted average, respectively did not affect our results.The study groups (healthy donors, HIV positive patients and their subgroups) were tested on significant differences in the magnitude of their responses by Student’s t-test. Additionally, we compared the data using the non-parameteric Wilcoxon Rank’s sum test. All p-values were not corrected for multiple comparisons. Longitudinal correlation We performed a dynamical correlation analysis as described elsewhere to test for correlation between the courses of specific antibody responses, the CD4+ T cell count or the plasma viral load in a single patient. ADDIN EN.CITE <EndNote><Cite><Author>Dubin</Author><Year>2005</Year><RecNum>45</RecNum><DisplayText>[3]</DisplayText><record><rec-number>45</rec-number><foreign-keys><key app="EN" db-id="rexwvprvkax297e2df4pxw9uzxapas909ze5">45</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Dubin, Joel A</author><author>Mueller, Hans-Georg</author></authors></contributors><titles><title>Dynamical Correlation for Multivariate Longitudinal Data</title><secondary-title>Journal of the American Statistical Association</secondary-title></titles><pages>872-881</pages><volume>100</volume><number>471</number><section>872</section><dates><year>2005</year></dates><urls><related-urls><url>;[3] This kind of correlation compares the trend rather than the magnitude of the measured variables.Analysis was performed using the R language of statistical computing (R Development Core Team 2008).References ADDIN EN.REFLIST 1 Slack E, Hapfelmeier S, Stecher B, et al. Innate and adaptive immunity cooperate flexibly to maintain host-microbiota mutualism. Science 2009;325:617-20.2 Galili U, Mandrell RE, Hamadeh RM, et al. Interaction between human natural anti-alpha-galactosyl immunoglobulin G and bacteria of the human flora. Infect Immun 1988;56:1730-7.3 Dubin JA, Mueller H-G. Dynamical Correlation for Multivariate Longitudinal Data. Journal of the American Statistical Association 2005;100:872-81. ................
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