The identification of brain-targeted autoantibodies in children with autism spectrum disorder (ASD) raises the possibility of autoimmune encephalopathy (AIE). Intravenous immunoglobulin (IVIG) is effective for AIE and for some children with ASD. Here, we present the largest case series of children with ASD treated with IVIG. Through an ASD clinic, we screened 82 children for AIE, 80 of them with ASD. IVIG was recommended for 49 (60%) with 31 (38%) receiving the treatment under our care team. The majority of parents (90%) reported some improvement with 71% reporting improvements in two or more symptoms. In a subset of patients, Aberrant Behavior Checklist (ABC) and/or Social Responsiveness Scale (SRS) were completed before and during IVIG treatment. Statistically significant improvement occurred in the SRS and ABC. The antidopamine D2L receptor antibody, the anti-tubulin antibody and the ratio of the antidopamine D2L to D1 receptor antibodies were related to changes in the ABC. The Cunningham Panel predicted SRS, ABC, parent-based treatment responses with good accuracy. Adverse effects were common (62%) but mostly limited to the infusion period. Only two (6%) patients discontinued IVIG because of adverse effects. Overall, our open-label case series provides support for the possibility that some children with ASD may benefit from IVIG. Given that adverse effects are not uncommon, IVIG treatment needs to be considered cautiously. We identified immune biomarkers in select IVIG responders but larger cohorts are needed to study immune biomarkers in more detail. Our small open-label exploratory trial provides evidence supporting a neuroimmune subgroup in patients with ASD. Although the standard-of-care for ASD is behavioral therapy, such therapy requires full-time engagement with one or several therapists for many years. In many cases, outcomes are suboptimal and/or incomplete2. Thus, medical therapies that can augment behavioral therapy are urgently needed.Autism spectrum disorder (ASD) is a behaviorally defined disorder, which now affects ~ 2% of children in the United States3. For example, a maternal immune response induced during gestation results in ASD-like behavior in offspring in the maternal immune activation (MIA) rodent5 and replacing the immune system using bone marrow transplant corrected many symptoms in a mouse model of Rett syndrome, a syndrome closely aligned with ASD6. In individuals with ASD, inflammatory cytokines are elevated in the blood7 and brain8 and microglia are active in the brain8. Children with ASD have autoantibodies to brain tissue such as myelin basic protein, serotonin receptors, brain endothelium, cerebellar tissue, and glutamic acid decarboxylase (GAD) as well as to non-brain tissue such as the folate receptor alpha (FRα)9 and mitochondria3. Children with autoantibodies are reported to have a more severe form of ASD15. The role of autoantibodies during gestation is exemplified by maternal antibodies to fetal brain that have high specificity for the development of ASD in the offspring16 with a relatively more severe phenotype17 and brain enlargement18.Recent studies suggest that ASD is associated with a variety of physiological abnormalities including immune system dysfunction19 and regulates catecholamine20 and glutamate21 neurotransmission. Abnormalities in CaMKII function has been linked to movement and neuropsychiatric disorders in children23 and genetic abnormalities in the CaMKII genes are linked to intellectual disability24. Furthermore, previous studies have linked PANDAS/PANS-associated biomarkers with behaviors commonly seen in ASD. For example, high levels of either the anti-lysoganglioside GM1 antibody or CaMKII are associated with tics and obsessive-compulsive disorder behavior25 and high antidopamine D2L to D1 antibody ratio is associated with irritability, a frequently targeted ASD symptom26.The overlap between ASD and Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infection (PANDAS) and Pediatric Acute-Onset Neuropsychiatric Syndrome (PANS) has been undergoing study by Cunningham et al. (in preparation) over the last several years. PANDAS/PANS-associated autoantibodies accelerate the function of the calcium calmodulin-dependent protein kinase II (CaMKII) enzyme, a multifunctional enzyme highly concentrated in the brain that controls neurotransmission and neuronal excitability33, voltage-gated potassium channel (VGKC)34, or N-methyl-d-aspartate (NMDA) receptor38 autoantibodies or without autoantibodies39. Seizure is the most common presenting symptom but children commonly also manifest motor, cognitive, behavioral, and/or psychiatric symptoms including aphasia35, declining academic functioning27, depression29, anxiety29, amnesia35, learning problems29, loss of social skills38, hallucinations34, agitation39, mood disorder34, behavioral changes39, decreased appetite36, irritability36, dysautonomia39, movement disorder35, peripheral neuropathy32, dystonia33, and insomnia39. Both GAD6540 and NMDA receptor autoantibodies38 have been associated with ASD with the NMDA receptor autoantibody being associated with regressive38 and late-onset37 ASD.Brain-targeted autoantibodies in an individual with central nervous system dysfunction point toward a diagnosis of an autoimmune encephalopathy (AIE). In children and adolescents AIE is most commonly diagnosed as non-neoplastic limbic encephalitis, resulting from GAD35 and case reports39 confirm that Intravenous immunoglobulin (IVIG) is useful for AIE in children and adolescents. IVIG has been shown to improve symptoms of ASD, including aberrant behavior41, speech and social interaction42, and ASD-related behaviors43. A small double-blind placebo-controlled study reported significant improvements in children with ASD receiving IVIG on the Aberrant Behavior Checklist (ABC) compared with those receiving placebo44. Another study demonstrated neurodevelopmental regression when discontinuing IVIG treatment41. In contrast, smaller case series did not find improvements in children with ASD with IVIG treatment46. As IVIG has been used to treat PANDAS/PANS symptoms in a placebo-controlled study47, case reports48, and case series49, IVIG could be a treatment for children with ASD with PANDAS/PANS symptoms.Case seriesGiven that children with ASD may manifest brain autoantibodies, we screened patients in an ASD clinic who did not respond to standard interventions for AIE. Selected patients with possible AIE were offered IVIG as a potential treatment. As part of our clinical protocol, families were asked to complete specific behavioral questionnaires, allowing the examination of the change in behavioral ratings for patients that received IVIG. In addition, clinical notes were analyzed to determine symptoms improvement, adverse effect, and treatment course. We also examined if immune biomarkers could predict response to IVIG treatment. Lastly, we provide several cases as illustrations of the treatment course with IVIG. This report provides insight to the possible utility of IVIG treatment in children with ASD as well as the potential limitations of this treatment.The Institutional Review Board at the University of Arkansas for Medical Sciences  approved the study. Parents of participants provided written informed consent. The study is registered as NCT02003170 on clinicaltrials.gov.All patients evaluated in our ASD multispecialty clinic for AIE were included in this study. The majority of the patients (80/82) were diagnosis with ASD, except for two females, one who was a sibling of a male with ASD and AIE who was treated for severe atypical learning disabilities and attention deficit and one with severe tics and behavioral dysregulation.53.ASD was diagnosed based upon one of the following criteria: (i) a gold-standard diagnostic instrument such as the Autism Diagnostic Observation Schedule and/or Autism Diagnostic Interview-Revised; (ii) the state of Arkansas diagnostic standard, defined as agreement of a physician, psychologist and speech therapist; and/or (iii) Diagnostic and Statistical Manual of Mental Disorders (DSM) diagnosis by a physician along with standardized validated questionnaires and diagnosis confirmation by the Principal Investigator (R.E.F.). We have validated that this criteria captures an accurate diagnosis of ASD in our previous studies by re-evaluating a portion of the participants with the ADI-R53.Table 40 and NMDA receptor54 autoantibodies have been associated with ASD, these two autoantibodies were measured specifically in most patients. The Cunningham panel (antidopamine D1 receptor (D1R), antidopamine D2L receptor (D2R), anti-lysoganglioside GM1, anti-tubulin, CaMKII; Moleculera Lab Inc, Oklahoma City, OK) was also performed as the symptoms associated with these autoantibodies overlap with behavioral symptoms of ASD. In some patients, the FRα autoantibody was measured because of its close association with ASD55. Some patients also had tests performed for markers of immune activation such as C3, C4, CH50, C-reactive protein, and erythrocyte sedimentation rate. Using findings from these laboratory tests along with clinical symptoms, the need for treatment of AIE with IVIG was considered, which is the focus of this report.The evaluation for AIE focused on detecting brain autoantibodies as measured by several panels. Paraneoplastic panel  measures antineuronal nuclear, anti-glial nuclear, Purkinje cytoplasmic, P/Q and N-type calcium, and VGKC antibodies as well as GAD65, AMPA, NMDA, and GABA-B receptor antibodies. Because GAD65M1 . Tubulin-, D1R-, and D2R-coated plates were washed three times with phosphate-buffered saline  containing 0.1% Tween . Lysoganglioside-coated plates were washed three times in PBS without Tween in all steps. Plates were blocked with 1% bovine serum albumin (ThermoFisher Scientific) in PBS for 60 minutes at 37 °C. Serum or cerebrospinal fluid (CSF) samples serially diluted in 1% BSA (in PBS) were added to washed plates, then incubated overnight at 4 °C. The next day, plates were washed as described above and primary IgG antibody binding was detected by adding 50 µL per well of diluted alkaline phosphatase-conjugated goat anti-human ƴ-chain-specific secondary antibody  and incubated for 60 min at 37 °C. Final dilution of secondary antibody was determined empirically for each antigen and validated for every new antibody lot. Plates were developed at 26 °C for 2 h ± 15 min with 50 µL per well of 1 mg/mL p-nitrophenylphosphatase (Sigma Aldrich) in 0.1 M diethanolamine buffer (pH 9.8). Optical density values were measured at 405 nm on an automated BioTek microplate reader  and corrected by blanks . All samples were assayed in duplicate and averaged. Duplicates not matching with < 20% variance were repeated. Titers represent the serum dilution at optical density of 0.1 at 405 nm after 2 h. Known positive and negative control samples were included on each plate to standardize and monitor assay performance29. Each new lot of all reagents and antibodies were validated using serum samples with known titers.Ninety-six-well microtiter plates  were coated with 50 μL of antigen in 100 mM carbonate/bicarbonate buffer (pH 9.6) and stored up to 2 weeks at 4 °C. Antigen coating concentrations were as follows: 10 µg/mL of purified tubulin , 10 µg/mL of dopamine D1 receptor , 10 µg/mL dopamine D2L receptor , and 20 µg/mL of lysoganglioside G15. Complete media contained 10% fetal bovine serum (ThermoFisher Scientific) and 1% penicillin–streptomycin antibiotic (ThermoFisher Scientific). Cellular extracts used in the CaMKII assay were centrifuged at 15,000 rpm for 10 min at 4 °C. Protein concentrations of the extracts were determined by Bradford assay using the Protein Assay Kit II .SK-N-SH human neuroblastoma cells obtained from American Type Culture Collection  were grown in complete F12-Dulbecco’s Modified Eagle Medium (ThermoFisher Scientific) as previously described15. In brief, SK-N-SH cells were plated in six-well plates at 2.5 million cells/well and incubated overnight in complete F12-DMEM, at 37 °C with 5% CO2. The next day, cells were serum starved for 30 minutes in serum-free F12 media with 2 mM CaCl2, 2 mM KCl, and 0.4 mM MgCl2, then stimulated for 30 minutes with patient sera or CSF diluted 1:100 in the same media or with media alone . Cells were harvested, centrifuged, solubilized in 0.165 mL of protein extraction buffer with protease inhibitors , and homogenized. Enzymatic activity was measured using the CaMKII assay system  per manufacturer’s instructions. In brief, 5 μL of cell lysate was incubated with 50 μM peptide substrate, buffers and ATP [γ-32P] (Perkin Elmer) for 2 minutes at 30 °C. Samples were spotted onto capture membranes and washed. Radioactivity retained on the membrane was measured by scintillation counter  and used to calculate specific activity of the CaMKII enzyme (pmoL/min/μg) as described in kit instructions. The protein concentration of each sample was used to standardize the CaMKII enzyme activity, and the percentage of specific activity of baseline  was calculated for each sample where the basal level was set at 100%. All samples were assayed in triplicate and results were averaged. Sera from patients with known high and low CaMKII activity and a basal control sample were included to standardize the assay.Assay for CaMKII activity was performed as previously describedThe first author, who was not involved in patient care, performed a qualitative–quantitative analysis of clinical notes. The senior author (R.E.F.), the primary treating physician, confirmed this analysis for accuracy. The notes were analyzed for specific symptoms mentioned in regards to improvements and worsening. Areas of improvement were quantified if they occurred in ≥ 10% of the participants. Symptom improvement was classified into eight non-exclusive categories  is a 65-item questionnaire that measures the severity of social skill deficits across five domains58, which has good concordance with the ADOS59. Caretakers are asked to complete these questionnaires prior to each clinical visit to assist in the evaluation of the change in symptoms at each visit. One important caveat is that caretakers were not required to fill out these questionnaires as declining did not ethically prevent their child from appropriate medical care.The ABC is a 58-item questionnaireα ≤ 0.05. All of the available questionnaire data were used for each participant. There was no imputation for missing data. Participants without questionnaire data both before and after the start of treatment were excluded. The Cohen’s d’ effect size was calculated for each statistical comparison.Statistical analyses were performed with SAS 9.4  and MATLAB . Graphs were produced using Excel version 14.0 . To determine whether any significant change occurred in behavior questionnaires with treatment an analysis of variance implemented as a mixed-model regression was utilized. The models included random effect of time (before/during treatment) and intercept to account for individual symptom level. The models tested the a priori hypothesis that a significant change in the outcome measure occurred with treatment and used a 26. We applied a logarithmic transformation with a base of 2 to the autoantibody titers as they increase in exponential steps. As many individuals completed multiple questionnaires both before and during the treatment period, we calculated the average total score of all questionnaires during treatment minus the average total score prior to treatment for each individual as a difference score. We then calculated the correlation between this difference score vs immune biomarkers.We sought to evaluate whether biomarkers of autoimmunity could predict treatment response. As the Cunningham panel was the only frequently abnormal panel, only the Cunningham panel was used to predict treatment response. We examined the relationship between the change in questionnaire score with IVIG treatment and the baseline components of the Cunningham panel. Only the total scores for the ABC and SRS questionnaires were examined to reduce excessive statistical comparisons. However, we also examined the relationship between the Anti-Dopamine D2L/D1 titer ratio and irritability subscale of the ABC, as this relationship was previously reportedWe then defined responders by examining the histogram of the change in the ABC and SRS scores to determine the distribution of responders. As the scale of the Cunningham panel components are quite different, in order to conduct calculations on the same scale across all components, as is standard is statistical linear models, were mean-centered at zero and normalized to have a standard deviation of unity. We then determined whether the components of the Cunningham panel could be used to predict which participants were responders and which where non-responders using Fisher Discriminant Analysis in MATLAB using in-house developed routine. Discriminant analysis creates a linear function that uses the input component  to calculate a score that represent whether a particular participant is more like a responder or non-responses. The statistical distribution of these Fisher Discriminant Analysis scores was estimated via Kernel Density Estimation and a threshold was determined which could optimally divide the scores calculated by the linear discriminant function for the participants into responders and non-responders . We report the accuracy, sensitivity, and specificity of the discriminant function.NMDA receptor autoantibodies were negative in all of the 34 patients in which it was tested. GAD65 titer elevation was found in three  < 0.02) of the 60 patients in whom it was tested. The PNP was positive for four (6%) of the 63 patients in which it was tested. For the PNP, two of the cases had VGCC autoantibodies  and two had striated muscle autoantibodies .22). In this way, the CaMKII elevation was used as a functional confirmation of the consequence of elevated autoantibodies in the ELISA. In the ELISA, the antineuronal autoantibodies were positive when anti-tubulin was ≥ 2000 titer, anti-lysoganglioside was ≥ 640 titer, anti-D1R was ≥ 4000 titer, and anti-D2R was ≥ 16,000 titer. Endpoint titers were determined at 0.092 cutoff and the titers were determined by routine ELISA as described above.The Cunningham panel was considered to be is positive only when one of the four autoantibody titers was positive in the ELISA AND at the same time autoantibody-mediated CaMKII activation was positive  of the 77 patients in which it was performed. In 67% of the 33 cases, the Cunningham panel was negative because of a normal CaMKII, whereas it was negative because of normal ELISA autoantibody titers in 30% of the cases and was normal for both ELISA autoantibodies and CaMKII in only one case.In addition to the aforementioned tests, patients with severe behavioral or medical symptoms including drug-resistance epilepsy and/or immunodeficiency were considered candidates for IVIG treatment. If the patient was previously non-responsive to IVIG treatment, alternative treatments were considered. Also, consideration was given to whether other treatments should be tried as an alternative to IVIG. The severity of the ASD and associated behavioral and other symptoms were a determinant when deciding whether a trial of IVIG was warranted.A trial of IVIG was recommended in 49 (60%) of the patients evaluated See Fig. . Thirty-Table All patients started on monthly dosing but 30% changed to a different dosing interval, with 17%, 6%, and 6% receiving the treatment at 3, 6, and 2 week intervals, respectively. In several cases, extending the treatment interval resulted in behavioral regression, requiring shortening the interval to restore treatment effectiveness (See Case #3 below).The majority of the parents reported improvements in communication and/or language with fewer reporting improvements in aberrant behavior Table . About aFor 21 and 20 of the treated children the SRS and ABC behavioral questionnaires were acquired both before and during IVIG treatment, respectively.p < 0.05, d’ = 0.58) and mannerisms  = 13.34, p < 0.001, d’ = 0.97) significantly improved with medium and large effect sizes, respectively. The total SRS score improved significantly with a medium effect size  = 4.40, p = 0.04, d’ = 0.57). Change in communication  = 2.60, p = 0.11, d’ = 0.43) and motivation  = 2.99, p = 0.09, d’ = 0.46) SRS subscales were borderline significant with medium effect sizes, whereas the change in the awareness subscale was not statistically significant  = 0.02, p = 0.89, d’ = 0.04).In Fig. p = 0.02, d’ = 0.64), lethargy   = 5.75, p = 0.02, d’ = 0.64), hyperactivity  = 4.63, p = 0.04, d’ = 0.57) and inappropriate speech  = 5.13, p = 0.02, d’ = 0.60) significantly improved with medium effect sizes. The total ABC score improved significantly with a large effect size  = 9.88, p = 0.003, d’ = 0.83). The improvement in the ABC stereotypy subscales was not significant and had a small effect size  = 1.90, p = 0.17, d’ = 0.36).In Fig. r = 0.55, p < 0.05; Fig. r = 0.49, p < 0.05; Fig. r = 0.64, p < 0.01; Fig. Biomarkers and behavioral questionnaires were available for 17 children for the SRS and 16 children for the ABC. Of the five biomarkers, the change in the total ABC score resulting from IVIG treatment was associated with the change in the anti-D2R antibody . Based on the distribution of scores a cutoff ≥ + 1 used to define a responder for the questionnaire scores. Using this criterion, five (25%) individuals were IVIG non-responders based on both questionnaires, three (15%) were IVIG responders based on the SRS only, four (20%) were IVIG responders based on the ABC only, and eight (40%) were responders based on both questionnaires. A criterion of two or more reported improvements in symptoms was used as a cutoff for parental reported improvement. Using these criteria, 52%, 60%, and 65% of patients were responders as defined by the SRS, ABC, and parental report. Thus, we believe that the criterion we used separated the groups well into responders and non-responders.The change in SRS scores is modest when it occurs, whereas the change in ABC scores can be rather marked when it occurs Fig. . Change The Cunningham panel predicted response to IVIG treatment with an accuracy of 81% with a sensitivity of 90% and a specificity of 67% based on the ABC scores; with an accuracy of 88% with a sensitivity of 100% and a specificity of 75% based on the SRS scores; and with an accuracy of 88% with a sensitivity of 100% and a specificity of 67% based on parental scores.The speed at which behavioral effects of the treatment arose and the duration of these effects were rather variable from patient to patient. However, ABC responders Fig.  demonstrOf the 31 patients who were initially treated with IVIG, 77% (24/31) continued with IVIG treatment long-term (> 1 year). The majority of these 24 patients, 88% (21/24), found continuing benefit from the treatment. Of the seven that stopped the IVIG treatment, 29% (2/7) stopped because of lack of insurance coverage, 29% (2/7) stopped because adverse effects, 14% (1/7) stopped in order to try a more intense immunosuppression treatment, 14% (1/7) stopped as a trial and requested to restart because of behavioral regression and 14% (1/7) was only prescribed one treatment. Only 6% (2/31) of the patients stopped the treatment because of lack of benefit and only 10% (3/13) who did not stop the treatment found limited continuing benefit.Sixty-five percent (20/31) of patients reported adverse effects, most commonly headaches and vomiting Table . Many adN,SD) 2.3 mM/L , nl < 1.3; 0.20 mM/L , nl < 0.08; 4.0 , nl < 2.5, respectively) and mitochondrial buccal swab demonstrating depressed citrate synthase (55%) and elevated complex I (178%) and IV (236%) function. A mitochondrial cocktail was started, including ubiquinol, l-carnitine, creatine monophosphate, niacin, pantothenic acid, pyridoxine, riboflavin, thiamine, cobalamin, and folinic acid. During the first treatment year he demonstrated improved adaptability, socialization, and ASD-related behavior. However, improvements in socialization plateaued and behavior worsened  loss in language ability as compared with the evaluation earlier in the year. At 4 years of age he was diagnosed with ASD. Gluten- and dairy-free diet reportedly improved focus and attention and speech, occupational, physical, and applied behavioral analysis therapies began. Family history was remarkable for learning disabilities in maternal family and developmental delays in the paternal family, but the mother, father, and sibling did not have a history of neurodevelopmental disorders. He was otherwise healthy.He was evaluated in our clinic at 4 years 6 months of age. On examination, he was active, cooperative, and anxious with very limited verbal communication skills but demonstrated eye contact. A mitochondrial and genetic workup  and overnight EEG were unremarkable. FRA, NMDA, and GAD65 antibodies were negative, however, PNP demonstrated an elevated N-Type Calcium-Channel antibody . A magnetic resonance imaging scan of the brain was normal and lumbar puncture demonstrated low protein .IVIG treatment was initiated at 1 g/kg/day for 2 days. Four weeks after the first IVIG infusion a marked improvement occurred in near constant chewing, repetitive movement, and severe sleep disruption. Five months after the first treatment, some ASD symptoms started to return, resulting in starting IVIG treatments on a monthly to every other month schedule. The N-type VGCC antibody dropped to 0.09 nmol/l just prior to the second IVIG treatment and then progressively further with additional treatments, reaching a low of 0.4 nmol/l for several months, and eventually dropping to 0.01 nmol/L Fig. . The ABCThis girl developed normally until about 3 years of age when she experienced periodic episodes of abrupt loss of previously acquired skills and developed other neuropsychiatric symptoms. The most significant of these regressions was at 4 years of age when she developed an abrupt onset of repetitive behaviors, tics, poor concentration, emotional liability, separation anxiety, urinary frequency and urgency, sleep disturbance, and aggressive behavior. She was diagnosed with ASD at age 8 years of age. At 9 years of age she developed partial complex seizures, incompletely controlled by lamotrigine. At 7 years of age, a Cunningham panel demonstrated a CaMKII (135; nl < 130; See Fig. In this study, we examined 82 children with neurodevelopmental disorders for biomarkers consistent with AIE and considered for immunomodulatory treatment for those with a clinical presentation and laboratory values consistent with AIE. Of those who underwent IVIG treatment using our care team, the majority had symptoms improvements as measured by standardized questionnaires. Although adverse effects were reported for the majority of patients, they were mostly limited to the time around the IVIG infusion, resulting in the great majority of patients considering the benefits outweighing any adverse effects.60. The child with ASD and the highest GAD65 titer also had type 1 diabetes, which is a disorder known to be associated with the GAD65 autoantibody31. This child was a responder to IVIG. Interestingly, GAD65 may be functionally disrupted in ASD in other ways besides an autoantibody mechanism. For example, GAD65 is downregulated in the prenatal valproic acid and MIA animal models of ASD65 and in post-mortem brain from individuals with ASD67, suggesting that it is a potentially important target of pathology in individuals with ASD.Interestingly, in our cohort of patients who presented to our ASD clinic, very few demonstrated autoantibodies usually associated with AIE in children. Interestingly, none of the patients was positive for NMDA receptor or VGKC autoantibodies. Only 5% of the patient cohort was positive for the GAD65 autoantibody with only one GAD65 titer within a clinically significant range. This low rate of positivity could explain the contradictory reports of the association of the GAD65 autoantibody with ASD as the rate of positivity may be very dependent on the sample of ASD children studied68 and cerebellar degeneration in childhood69. The child positive for the N-type VGCC demonstrated an abrupt onset of sleep disturbance, repetitive behavior, and chewing on objects in addition to language and social regression at the onset of this disorder. IVIG treatment significantly and quickly improved these symptoms. Interestingly, like the GAD enzyme, ASD has been associated with non-autoimmune disruption in calcium-channel function70. Thus, some autoantibodies in individuals with ASD may merely represent one mechanism that can disrupt common pathophysiological disturbances leading to ASD.This is the first reported association of VGCC autoantibodies with ASD. The VGCC is associated with a wide range of cognitive and motor disturbances44 and social interactions42. Of the six studies using IVIG in the treatment of ASD, three open-label studies, totaling 63 patients43 and one double-blind placebo-controlled study totaling 12 patients44 demonstrated a benefit, whereas two smaller open-label studies, totaling 15 patients46, did not show benefit. Two of the studies that demonstrated benefit selected individuals with immune system abnormalities, like the current study43. In contrast, the two open-label studies that did not demonstrate benefit did not select for immune abnormalities46. In addition, all previous studies used a lower dose of IVIG, usually 0.4 g/kg monthly. Some have observed better response with higher doses of IVIG71 potentially explaining the high rate of response in our study. Clearly more research is needed to better understand which subset of children with ASD can best benefit from IVIG as well as the optimal dose and interval for the treatment.Overall, the majority of parents of the patients treated with IVIG found the treatment beneficial and believed that the benefits outweighed any adverse effects. Close observation was important to tailor the dose and frequency of the treatment and minimize adverse effects. Consistent with previous literature, IVIG treatment in this study improved scores on the ABC22 and PANDAS26.The majority of the ASD patients who had autoantibodies demonstrated elevations in autoantibodies measured by the Cunningham panel along with an elevation in the activation of CaMKII. Our analysis suggests that two of the autoantibodies, the anti-tubulin and anti-D2R were associated with responsiveness to IVIG treatment, suggesting that these could be biomarkers to select individuals who might benefit most from IVIG treatment. It should be noted that the participants were selected based on an elevated CaMKII, so this biomarker may have been artificially fixed with a limited dynamic range because of our patient selection process, thereby decreasing its ability to be a predictive biomarker. As the ABC was most closely associated with immune biomarkers, it may be that the autoantibodies identified as predictive of response to IVIG may be closely associated with modulation of behavior as has been suggested in Sydenham choreaThe limitations of this study include the small sample size and the questionnaire-based outcome measures. Furthermore, this study was not a systematic clinical trial with comprehensive measurements of behavior, cognition, and language. Thus, further studies are needed to better identify which specific symptoms are targeted by IVIG treatment as well as the most-sensitive biomarkers to identify which patients are most likely to respond to IVIG treatment. Despite these limitations, our study verifies previous reports that suggest that IVIG may be useful in individuals with ASD who demonstrate biomarkers of immune abnormalities.Table S1


Hermetia illucens L.) and yellow mealworm larvae  on growth performance, nutrient digestibility, blood parameters, intestinal morphology and health of growing rabbits.The present work aimed at evaluating the effect of the dietary replacement of soybean oil (S) by two types of insect fats extracted from black soldier fly larvae  were allotted to five dietary treatments (40 rabbits/group): a control diet (C) containing 1.5% of soybean oil and four experimental diets where soybean oil was partially (50%) or totally (100%) substituted by H (H50 and H100) or T (T50 and T100) fats. Total tract digestibility was evaluated on 12 rabbits per treatment. The growth trial lasted 41 d and, at slaughtering (78 days old), blood samples were collected from 15 rabbits per treatment, morphometric analyses were performed on duodenum, jejunum and ileum mucosa, and samples of liver, spleen and kidney were submitted to histological evaluation.No difference was observed between the control and the experimental groups fed insect fats in terms of performance, morbidity, mortality and blood variables. The addition of H and T fats did not influence apparent digestibility coefficients of dry matter, protein, ether extract, fibre fractions and gross energy. Gut morphometric indices and organ histopathology were not affected by dietary inclusion of H and T fats.H and T fats are suitable sources of lipid in rabbit diets to replace soybean oil without any detrimental effect on growth performance, apparent digestibility, gut mucosa traits and health.The online version of this article (10.1186/s40104-018-0309-2) contains supplementary material, which is available to authorized users. Several researches focused on the influence of the dietary supplementation of different fat sources such as animal oil or plant oilseeds –3 on perHermetia illucens L.) and yellow mealworm  [The 60%–70% increase of animal products consumption expected by 2050 has pushed research to investigate new raw materials for animal feeds . In thisitor L.) , 10.Nevertheless, insects have different lipid content  and FA profile related to the species and the rearing substrate , 11, 12.Hermetia illucens is a worldwide distributed Diptera which is reared for its ability to convert low value substrates into valuable products [Tenebrio molitor is a Coleoptera which larvae, considered as pests of stored grains, contain from 15% to 45% DM of fat, mostly palmitic (C14:0), oleic (C18:1 n-9) and linoleic (C18:2 n-6) acids [Concerning the insect species mentioned above, products , 18, andproducts . Tenebri C18:2 n- acids [66) acids .Tenebrio molitor or Zophobas morio fat (5% of dietary inclusion) in broiler diets without adverse consequences on growth performance and nutrient digestibility.So far, the use of insect lipids in animal feeding has received little attention. Researchers investigated the effects of the dietary inclusion of H fat in partial or total replacement of soybean oil (S) without negative effects on performance and carcass traits of broiler chickens , 20. Li As far as rabbits are concerned, to the best of our knowledge only two old researches have been carried out using insect meal (silkworm) replacing soybean meal , 22 and The Ethical Committee of the University of Turin  approved the experimental protocol . The trial was carried out at the experimental rabbit facility of the Department of Agricultural, Forest, and Food Sciences .An experimental control diet (C) containing 1.5% soybean oil and formulated to satisfy growing rabbits requirements  was testAfter weaning at 36 days of age, 200 commercial crossbred rabbits  purchased from a commercial farm , 1051 ± 138 g live weight (LW), were randomly allocated in individual wire-net cages (41 cm × 30 cm × 28 cm height). Forty rabbits, homogeneous by LW and variability, were assigned to each dietary treatment. The rabbitry had automatic control of environmental temperature (22 ± 2 °C) and photoperiod (16L:8D). The rabbits were fed ad libitum and had free access to clean drinking water during the whole trial that lasted 41 d, until 77 days of age.At 78 days of age, rabbits were slaughtered in an experimental slaughterhouse. Data of dressing percentage, carcass traits and meat quality, and chemical composition were determined but not reported in this paper.During the trial, mortality and morbidity were controlled daily by the same observer according to Gidenne et al. . LW and The apparent digestibility coefficient (ADC) and the nutritive value of experimental diets were measured during an in vivo digestibility assay performed according to the European standardised method . The triDiets and faeces were analysed to determine the contents of DM (method 934.01), ash (method 967.05), crude protein (CP - method 2001.11), and starch  using AOAC methods  followinThe FA composition of insect lipids and feeds was determined according to Trocino et al. . The fatg for 15 min and frozen at − 80 °C until analysis. The total proteins (TP) were quantified by the “biuret method” . Alanino-aminotransferase (ALT), aspartate-aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), triglycerides, cholesterol, calcium, phosphorus, iron, uric acid, urea, lactate dehydrogenase (LDH) and creatinine serum concentrations were analysed with enzymatic methods using the Screen Master Touch automated instrument .At slaughtering, blood samples were collected from 15 rabbits per treatment at the moment of jugular exsanguination and put into 2.5 mL serum-separating tubes. The tubes without anticoagulant were left to clot in a standing position at room temperature for approximately two hours to obtain serum. The serum was separated by means of centrifugation at 700×The same 15 rabbits per treatment used for serum biochemistry determinations were used for anatomo-pathological investigations. Segments (approximately 5 cm in length) of duodenum (after the pylorus), jejunum (middle portion) and ileum  were excised and flushed with 0.9% saline to remove all the intestinal content. Samples of liver, spleen and kidney were also collected. The collected samples were fixed in 10% buffered formalin solution and submitted to morphometric analysis (intestine tracts) and histopathological examination (other organs). The tissues were routinely embedded in paraffin wax blocks, sectioned at 5 μm thickness and mounted on glass slides. Intestinal sections were submitted to haematoxylin-eosin (HE) staining, with a total of five serial sections prepared for each intestinal segment. The same slide among the serial sections was examined by light microscopy and captured with a Nikon DS-Fi1 digital camera coupled to a Zeiss Axiophot microscope using a 2.5× objective lens. NIS-Elements F software was used for image capturing. Morphometric analysis was performed by Image®-Pro Plus software. The evaluated morphometric indices were: villus height , crypt depth  and the villus height to crypt depth ratio , mild (score = 1), moderate (score = 2) and severe (score = 3). The following histopathological alterations were evaluated: inflammatory infiltrates and degenerative changes in liver and kidney and white pulp hyperplasia and depletion in spleen. All slides were assessed blinded by three observers and the discordant cases were reviewed at a multi-head microscope until a consensus was reached.The statistical analyses were performed using the IBM SPSS software package . Shapiro-Wilk’s test established normality or non-normality of distribution. One-way ANOVA was used to evaluate the effect of experimental diets on growth performance, apparent digestibility coefficients, serum biochemical parameters and intestinal morphometric indices. The assumption of equal variances was assessed by Levene’s homogeneity of variance test. Mortality rate was analysed by Chi-square test, using the C group as the reference. Differences amongst groups were evaluated by the Duncan’s test. Histopathological scores were analysed by Kruskal-Wallis test (post-hoc test: Dunn’s Multiple Comparison test).P ≤ 0.05 and a statistical trend was considered for 0.05 < P ≤ 0.10. The results are presented as the means and standard error of the means (SEM).For all statistical analyses significance was declared at Diets were comparable in terms of main crude protein (on average 16.6% DM) and gross energy contents (18.6 MJ/kg DM), despite some differences in aNDF and ADF contents (Table n-9) and MUFA rates were the highest in T fat and T diets; whereas, linoleic acid (C18:2 n-6) and PUFA dominated the FA profile of S and C diets.The substitution of S with insect fats modified the FA profile of the lipids in the diets Table . In factTable P = 0.08).The ADC are reported in Table P > 0.10) the serum biochemical parameters of the rabbits (Table P ≤ 0.10).The different dietary H or T fat inclusion levels did not influence .Histopathological alterations were present in all the organs of all rabbits regardless from the dietary treatments Table . MultifoOnly few papers investigated the effects of insect oils in poultry feeds , 17, 20 Zophobas morio fats in total substitution of S in a 28-day trial did not affect the growth and feed efficiency of broiler chickens [At the end of the growing period, no statistical differences among groups were reported for the growth performance traits. During the trial, the rabbits were not treated with antibiotics and the mortality was low and not influenced by dietary treatment. The similar feed intake indicated that 0.75% and 1.50% inclusion levels of both insect fats were acceptable to growing rabbits. A preference test where poultry were free to choose between diets containing H or S fats did not reveal any difference, confirming a good acceptability of both insect lipid sources . Our datchickens .Cyprinus carpio var. Jian) and reported no differences on growth performance and nutrient utilization. Recently, Dumas et al. [As far as fish are concerned, Li et al.  studied s et al.  evaluateOther researches using different  oils and fats in rabbit diets did not find significant differences in growth performance, indicating that rabbit can digest and metabolize different lipid sources, at least until 3% of inclusion level  and KowaTo go in deep on the evaluation of dietary treatments on nutrient digestibility, in the present trial DM and energy digestibility coefficients were high and similar in all treatments indicating that both insect lipid sources are suitable for rabbit feeding. As the digestibility of EE is concerned, the mean value was consistent with literature data. Differences may depend on the type of fat source, their unsaturation degree as well as the level of structural lipids linked to cell walls , 44, 45.In the contrary, Martins et al.  reportedIn the current trial, the values of the different traits lay within the normal range for rabbits –49. ThisIndeed, also in broiler chickens, Schiavone et al. , 20 showSince both gut morphology and growth performance were unaffected in H and T groups of the current research, insect lipids utilization did not negatively influence gut development and functionality. Consistently with the results of the present study, Schiavone et al.  observedDietary H or T fat inclusion did not affect either the development or the severity of the histopathological alterations in the rabbits of the current research. Degenerative and inflammatory changes of liver and kidney are usually observed in toxic diseases  or toxicThe obtained results showed that H and T fats can be used as partial or total substitute of soybean oil until 1.5% of the diet in rabbit feeding without impacting growth performance, nutrient digestibility, serum biochemical traits and gut development. These findings suggest that H and T fats is a suitable ingredient for rabbit diets. However, further studies should be performed to establish the most effective source and to determine the optimal inclusion level of insect lipids for rabbit feeding.Additional file 1:Morphometric evaluation of the jejunum segment of the rabbits. Morphometric measurements of the villus height (Vh) and the crypt depth (Cd). H100 group, 2.5× haematoxylin-eosin stain. (TIF 7150 kb)Additional file 2Histopathological findings of the rabbits. (A) HI50 group. A normal liver is observed. 10× haematoxylin-eosin stain. (B) H100 group. Liver, periportal zone. Moderate and multifocal vacuolar degeneration of the hepatocytes (arrowheads), as well as mild and multifocal lymphoplasmacytic inflammation (arrows), are identified. 10× haematoxylin-eosin stain. (TIF 5050 kb)


Malaria epidemiological and immunological data suggest that parasite tolerance wanes in the absence of continuous exposure to the parasite, potentially enhancing pathogenesis. The expansion of control interventions and elimination campaigns raises the necessity to better understand the host factors leading to susceptibility or tolerance that are affected by rapid changes in malaria transmission intensity (MTI). Mediators of cellular immune responses are responsible for the symptoms and pathological alterations during disease and are expected to change rapidly upon malaria exposure or cessation.Plasmodium falciparum (markers of exposure) was explored. The effects of age, sex, neighbourhood and parasitaemia on analyte levels and their interactions with year were also assessed.The plasma concentrations of 30 cytokine, chemokine and growth factors in individuals of all ages from a malaria endemic area of southern Mozambique were compared between 2 years of different MTI: 2010  and 2013 . The effect of the year on the correlations between cytokines, chemokines and growth factors and IgGs to P. falciparum-infected and uninfected subjects. Age and neighbourhood showed an effect on analyte concentrations.An inverse correlation of several cellular immune mediators with malarial antibodies in 2013, and a lack of correlation or even a positive correlation in 2010 were observed. Most cytokines, chemokines and growth factors, regardless of their immune function, had higher concentrations in 2010 compared with 2013 in The results show a different regulation of the cellular immune response in 2010 vs 2013 which could be related to a loss of immune-tolerance after a decline in MTI in 2010 and previous years, and a rapid re-establishment of tolerance as a consequence of more continuous exposure as MTI began increasing in 2012. Cellular immune mediators warrant further investigation as possible surrogates of MTI-associated host susceptibility or tolerance. The burden of malaria disease has experienced significant changes in endemic areas in the 21st century. Between 2000 and 2015, an expansion of malaria interventions helped to reduce malaria incidence by 37% globally, and by 42% in Africa. However, between 2015 and 2017 no significant progress was made, with an estimated 219 million cases and 435 000 related deaths in 2017 [Plasmodium parasite dynamics. Thus, the expansion of control interventions and elimination campaigns raises the necessity to better understand the host factors affected by rapid changes in MTI.Geographical and temporal changes in malaria transmission intensity (MTI) can affect disease burden and Plasmodium falciparum infection. NAI is suggested to be comprised of two main components: (i) an anti-parasite component, resulting in control of parasite replication and parasite clearance, which takes years to be acquired and is never sterilizing [In endemic areas of Africa, naturally acquired immunity (NAI) to malaria is developed with age and exposure to rilizing , 3; and rilizing , 5.Tolerance is a less understood phenomenon. From the immunological perspective, it is defined as any endogenous mechanism by which a potentially injurious immune response is prevented, suppressed, or shifted to a non-injurious response . In malaEpidemiological and immunological data suggest that anti-parasite immunity and tolerance wane in the absence of continuous exposure to the parasite , and chaP. falciparum appeared to be reduced [P. falciparum would result in a lesser development of NAI in children, and a possible partial loss of previous immunity and tolerance in the older population if exposure was very low or discontinued, which may enhance malaria pathogenesis.Temporally, immune adults who migrate to non-endemic areas are at significant risk of contracting malaria upon return to an endemic area, even increased susceptibility to severe malaria . Previou reduced . Thus, aP. falciparum-specific cytokine responses have been associated with protection against clinical malaria, including interferon gamma (IFN-γ) [Cytokines, chemokines and growth factors mediate cellular responses and are responsible for the symptoms and pathological alterations during disease. The outcome of infection depends on the regulation of pro-inflammatory and anti-inflammatory responses, leading to protection or immunopathology . Thus, b (IFN-γ) –17, inte (IFN-γ) , 19. Oth (IFN-γ) , 20. Ser (IFN-γ) .P. falciparum infected but also in uninfected subjects. This could be indicative of a loss of immune-tolerance after years of decline in MTI, and a re-establishment of the tolerance as a consequence of a more continuous exposure due to a rise in MTI starting before 2013. The possibility of cytokines as surrogates of loss of parasite tolerance upon changes in MTI deserves further investigation.In this study, changes in cellular immune mediator profiles in 2 years of different MTIs were assessed in individuals of all ages from a malaria endemic area of southern Mozambique. To address this, a comprehensive panel of 30 cytokines, chemokines and growth factors, several of them known to vary upon malaria infection and/or exposure , 24, 25,P. falciparum malaria is perennial and of moderate intensity, with two different seasons, a warm and rainy season from November to April, and a cool and dry season the rest of the year [http://chg.geog.ucsb.edu/data/chirps/). The cumulative rainfall levels between the months of September 2009 and January 2010 was 310.76 mm, and doubled to 625.26 mm between the months of September 2012 and January 2013. Individuals of all ages were selected by random age-stratified sampling from the demographic surveillance system census and were invited to participate in the study. Weighted sampling was done according to the following age groups: Infants < 1 year; 1 ≤ 2 years; 2 ≤ 3 years; 3 ≤ 4 years; 4 ≤ 5 years; 5 ≤ 10 years; 10 ≤ 20 years; 20 ≤ 40 years; 40 ≤ 60 years; ≥ 60 years. Participants were recruited from six different neighbourhoods: Malavele, Manhiça and Maragra, historically considered of low MTI; and Ilha Josina, Palmeira and Taninga, of moderate MTI [Plasmodium parasitaemia according to standard, quality-controlled procedures [This study was developed in the context of yearly cross-sectional surveys performed to monitor changes in malaria burden and MTI in the Manhiça District, Maputo Province, in southern Mozambique, which started in 2010 and are still on going. The characteristics of the study area have been described in detail elsewhere . The clithe year . The crorate MTI . A standocedures .P. falciparum parasites in blood detected qPCR, together with fever or reported fever during the previous 24 h. Febrile infections with parasites also detected by microscopy were considered clinical malarias. Anemia was defined as haemoglobin < 11 g/dL. Participants presenting with parasitaemia, anaemia, fever or history of fever in the previous 24 h were treated according to standard procedures. All malaria infections were treated with the first-line anti-malarial treatment (Coartem), and anaemia cases received ferrous sulphate, according to national guidelines. Participants presenting signs/symptoms of severity were transferred to the Manhiça District Hospital.Haemoglobin was measured using HemoCue portable devices . A blood aliquot was collected in an EDTA microtainer and plasma separated by centrifugation and cryopreserved at − 80 °C for immunological analyses. Blood was also collected onto filter paper for IgG quantification by ELISA , and forPlasma samples analysed in this study were from 377 participants from the surveys performed in 2010  and 2013 . Years 2010 and 2013 were defined as lower and higher MTI periods, respectively, based on the trends reported in Mozambique, with a decrease in malaria prevalence from 2007 to 2011, and an increase since 2012 , 33.All plasmas from infected individuals (161 participants qPCR positive) and the plasmas from 216 non-infected participants (qPCR negative) were analysed separately to assess cytokine profiles in 2010 and 2013 during an infection and at baseline, respectively. The plasmas from non-infected individuals were randomly selected from the surveys, balancing between years and stratifying by specific age groups , with the last two groups enriched to have a sample size powered to address older age effects.The Cytokine Human Magnetic 30-Plex Panel from Life Technologies™ was used to measure the concentrations (pg/mL) of the following cytokines, chemokines and growth factors in plasma: epidermal growth factor (EGF), fibroblast growth factor (FGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), tumour necrosis factor (TNF), interferon (IFN)-α, IFN-γ, interleukin (IL)-1RA, IL-1β, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p40/p70), IL-13, IL-15, IL-17, IFN-γ induced protein (IP-10), monocyte chemoattractant protein (MCP-1), monokine induced by IFN-γ (MIG), macrophage inflammatory protein (MIP)-1α, MIP-1β and regulated on activation normal T cell expressed and secreted (RANTES) and eotaxin. This panel has been used in previous studies showing several analytes varying upon malaria infection and/or exposure , 24, 25.® 100/200 instrument and analysed in xPONENT® software 3.1. The concentration of each analyte was obtained by interpolating the median fluorescent intensity (MFI) (after blank MFI subtraction) to a 5-parameter logistic regression curve automatically calculated by xPONENT® software. Any value below the lower limit of detection (mean of blanks + 2 standard deviations) was assigned half the expected concentration at the low limit of quantification for that analyte.Twenty-five microlitres of all plasmas were tested in single replicates applying a modification of the manufacturer’s protocol that implies using half the volume of each reagent except for the washing buffer ; this moAntibody data obtained in a separate study of seroconversion rates performed in the context of the Manhiça cross-sectional surveys were analysed in relation to the cytokine data. Briefly, 2.5 mm disks of dried blood (≈ 1.5 µL of blood) were cut from filter paper spots and incubated overnight at room temperature (RT) with 150 mL of PBS/0.05% Tween 20/0.01% sodium azide (w/v) .42) (3D7 strain) or apical membrane protein-1 (AMA-1) (FVO strain) produced at the Walter Reed Army Institute of Research , at 0.5 mg/mL of carbonate buffer. PBS with 0.05% Tween (PBS-T) was used to wash plates between incubations. Plates were blocked with 1% skimmed milk powder  in PBS-T (blocking buffer) for 3 h at RT. Reconstituted antibodies were transferred to the ELISA plates  and incubated overnight at 4 °C. HRP-conjugated rabbit anti-human IgG  was applied at 1/5000 in PBS-T and incubated for 3 h at RT. OPD development substrate was applied and incubated in the dark at RT for 20–25 min, stopping development with the addition of 2 M H2SO4. Plates were read on Bio-Tek ELx 50 plate reader using KC Junior software package . Normalized optical density (OD) was calculated as the mean background-adjusted OD divided by the mean of a hyperimmune plasma. The same standard dilution was used for normalizing each plate in the study. Test samples were assayed in duplicate and included in the analysis if the coefficient of variation was less than 50% for all values greater than 0.1 OD.Reconstituted sera were stored at − 20 °C until use. The ELISA assay was performed as previously described . High-bi10 transformed for further analysis.Plasmas from malaria-infected and non-infected volunteers were analysed separately. The studied population was categorized into 6 age groups  according to commonly observed immunological patterns. Demographic continuous variables were analysed using the non-parametric Wilcoxon rank-sum test. Comparisons between groups for categorical variables were done using Fisher’s exact test. Concentrations of cytokines, chemokines, growth factors, parasite densities and IgG levels (OD) were logComparison of cellular analyte concentrations between two or more groups was performed through Wilcoxon rank-sum test or Kruskall–wallis test, respectively. Comparisons of IgG levels between years were performed by Wilcoxon rank sum test with continuity correction for all age groups together or into each age category separately. Trends of IgG levels along age groups were assessed by Spearman correlations (p-trend). The correlations of IgG levels and analyte concentrations were assessed with univariable linear models, with IgG levels as independent variable, separate for each analyte. The effects of year, age group, neighbourhood, sex and parasite density (predictors) on analyte levels (outcome) were assessed through univariable and multivariable (adjusted models) linear regressions, separate for each analyte. Interaction tests were performed to determine if there was an interaction of age group, neighbourhood, sex, parasite density and antibody levels with year on the analyte levels. When interactions were statistically significant, a stratified analysis was performed.P-values were adjusted for multiple testing to control the false discovery rate using the Benjamini–Hochberg approach in each study endpoint separately  except for the comparison of antibody levels between years into age categories for which the Holm method was used. In all endpoints evaluating cytokine associations of this exploratory study, the Benjamini–Hochberg approach was used because it has more power than family-wise-error rate methods, such as Bonferroni or Holm, and allows identifying as many significant associations as possible while incurring a relatively low proportion of false positives [ggplot2 package [compare Groups package was used for computing p-trend values [fmsb package [radarchart function. The reshape and dyplr packages were used for data manipulation purposes [ReporteRs package for exporting data tables in Microsoft Word documents [All p-values were considered statistically significant when < 0.05. ositives . For theositives . The ggp package  was usedd values . The fms package  was usedpurposes  and the ocuments .P. falciparum infection in 2010 and 2013 were 1.1% and 5.2% by microscopy, and 12.6% and 23.9% by qPCR, respectively; and the numbers of febrile infections were 15 and 43, being 3 and 18 clinical malarias, respectively  individuals were tested by microscopy and 970 (99%) by qPCR; while 944 (96.3%) and 808 (82.4%) individuals were tested by microscopy and qPCR, respectively, in 2013. Weighted prevalence of 42 were higher in 2013 compared to 2010 (p < 0.001 for both IgGs) mirroring the MTI trends between 2010 and 2013, and increased with age within each cross-sectional , reflecting the continuous exposure to P. falciparum infection  and parasitaemia [Recent studies suggest that repeated exposure to olerance , which molerance , αβ T ceolerance –52, B ceolerance , and myeolerance  that shoolerance , 56. A rsitaemia . Accordisitaemia .P. falciparum and an increase of malaria adverse consequences after the decline of MTI [P. falciparum infection at the time of the survey, suggesting that the rate of previous exposures could shape the basal immunological status. This difference in the basal immune system agrees with the observation by Tran et al. [Plasmodium infection. This study also suggests that previous exposure to malaria, and possibly other pathogens or commensals, shape the basal immune system.Another study showed diminished cytokine response with age in children, in part probably due to increased exposure, which could also be indicative of tolerogenic mechanisms . A decree of MTI . Interesn et al.  of a marP. falciparum [Plasmodium species susceptibility [In the same line, a previous study by our group in semi-immune African adults, migrants and European travellers, showed that naïve adults had stronger cytokine responses upon infection than semi-immune adults; and that migrants, in the absence of continuous exposure, presented higher concentrations of cytokines and chemokines than semi-immune individuals, but lower than individuals with a first infection . There, lciparum . Similarlciparum , 47, 62.tibility , 64 and tibility , being dtibility . This imDespite the lower cellular immune mediators observed in 2013, infected individuals showed higher levels of several cytokines and chemokines compared to the uninfected ones, differences that were not observed in 2010; among them, the pro-inflammatory cytokines IFN-α, IL-1β and IL-6, the chemokines IL-8, MCP-1, MIP-1α and MIP-1β and the growth factor VEGF. This may be related to the lower basal levels of cellular immune mediators in 2013 compared to 2010. In 2010, basal levels of cellular mediators were so high that infections might have not been able to induce further increases; whereas in 2013, cytokine, chemokine and growth factor basal levels were low and infections increased them significantly, though still below the basal levels from 2010. This observation supports the hypothesis of a more controlled, non-harmful cellular immune response upon re-infection in 2013.Despite the overall decrease in cellular immune mediators in 2013, a higher prevalence of clinical malaria (12%) was registered compared to 2010 (3%) among the infected individuals, similar to the prevalence in the surveys (18/162 vs 3/106). However, the total number of clinical malaria cases was low, and clinical malaria depends not only on tolerogenic responses but also on anti-parasite immunity. During lower MTI periods there is a delay in the acquisition of immunity in younger children and a reduction in premunition in older children and adults; thus, in the context of a sudden increase of MTI, anti-parasite immunity may be acquired slower than tolerogenic responses. It is likely that a higher number of clinical cases would have been observed in 2013 had some level of tolerance not been developed.As expected, among the infected individuals the level of parasitaemia affected several analytes, some of them similar to previous reports , 66. IntH1 cytokines IL-2, IL-12 and IL-2R and the TH1 related IL-15 decreased continuously with age; while eotaxin, IP-10 and the TH2 cytokines IL-13, IL-4 and IL-5 presented a U-shape, i.e. higher levels in children and the elderly and lower levels in young adults. Previous studies have shown that some cytokines increase in the elderly [Age-related differences in immunity are suggested to also explain the different susceptibility to malaria disease in children vs adults  independ elderly , while o elderly , 73, but elderly .Previous studies have also shown that the severity of malaria infection differs between males and females , with meP. falciparum IgG levels in 2013 compared to 2010 across all age groups and negative correlations with cellular immune mediators suggests that P. falciparum exposure could be driving the changes described. Future studies should have a longitudinal design and comprise different MTI periods to address the role of cellular immune mediators as surrogates of MTI and tolerance/susceptibility to malaria. In addition, controlling for other factors influencing the basal immune system would also be important. Understanding the mechanisms of longevity of immunity and tolerance may help in developing approaches to promote long-lasting anti-disease immunity, even after sustained interrupted exposure, which could be used in areas of low MTI and in elimination campaigns.There are some limitations in this study that could have affected the results. First of all, the different storage times for plasma samples collected in 2010 vs samples collected in 2013, which may have differently affected the cytokine concentrations. However, storage time tends to decrease the concentration of most of the cytokines . TherefoP. falciparum infected and non-infected individuals, which could be related to a loss of immune tolerance when MTI declines and a rapid re-establishment of immune tolerance after an increase in P. falciparum exposure. This finding warrants further investigations on plasma cellular immune mediators as potential surrogate candidates of MTI and the associated host susceptibility and tolerance.Cytokine, chemokine and growth factor profiles varied between years of different MTIs in Additional file 1: Table S1. Monthly mean rainfall in the study area in the 5 months previous to the 2010 and 2013 surveys. Table S2. Effect of infection on analyte concentrations and the interactions with year. Table S3. Interaction of age with year on analyte levels in infected and uninfected individuals. Table S4. Effect of sex on analyte concentrations and the interactions with year in infected and uninfected individuals. Table S5. Interaction of neighborhood with year on analyte levels in infected and uninfected individuals.Additional file 2. Differences in cellular immune mediator concentrations between 2010 (lower MTI) and 2013 (higher MTI) in P. falciparum-infected subjects. Box plots representing the median and interquartile range of each analyte concentration (log10 pg/mL) in infected subjects recruited in 2010 and in 2013. Levels between both years were compared by Wilcoxon rank-sum test and p-values were adjusted for multiple testing by Benjamini–Hochberg approach.Additional file 3. Differences in cellular immune mediator concentrations between 2010 (lower MTI) and 2013 (higher MTI) in uninfected subjects. Box plots representing the median and interquartile range of each analyte concentration (log10 pg/mL) in uninfected subjects recruited in 2010 and in 2013. Levels between both years were compared by Wilcoxon rank-sum test and p-values were adjusted for multiple testing by the Benjamini–Hochberg approach.Additional file 4. Correlations of VEGF, MCP-1 and IFN-α with P. falciparum antibody levels stratified by year. Data are presented in scatter plots with trend lines, with analytes concentrations in the Y axes and antibody levels in the X axes. R2 and p-values were obtained through linear regression models. Only analytes that had a statistically significant interaction with year for the correlation with antibodies are shown.Additional file 5. Cellular immune mediator concentrations in 2010 (low MTI) and 2013 (high MTI) stratified by age group. Box plots representing the median and interquartile range of analytes concentrations (log10 pg/mL) in P. falciparum infected (a) and uninfected (b) subjects. Only analytes in which age and year had a significant p-value for the interaction test (before correcting for multiple testing) are shown.Additional file 6. Cellular immune mediator concentrations in 2010 (low MTI) and 2013 (high MTI) stratified by sex. Box plots representing the median and interquartile range of analyte concentrations (log10 pg/mL) in P. falciparum infected (a) and uninfected (b) subjects. Only analytes in which sex and year had a significant p-value for the interaction test (before correcting for multiple testing) are shown.Additional file 7. Differences in cellular immune mediator concentrations between areas in P. falciparum infected subjects. Box plots representing the median and interquartile range of each analyte concentration (log10 pg/mL) in infected subjects stratified by neighborhood. Levels between areas have been compared by Kruskal–Wallis test.Additional file 8. Effect of parasitema on IL-10 and GM-CSF concentrations stratified by year. Scatter plots with trend line representing the distribution of analytes concentration by parasitemia stratified by year. Only analytes in which parasitemia and year had a significant p-value for the interaction test (before correcting for multiple testing) are shown.


Lawsonia intracellularis is among the most important enteric pathogens of swine, and it can also infect other mammalian species. Much is still unknown regarding its pathogenesis and the host response, especially at the site of infection. In this study, we uncovered several novel genes and pathways associated with infection. Differentially expressed transcripts, in addition to histological changes in infected tissue, revealed striking similarities between L. intracellularis infection and cellular proliferation mechanisms described in some cancers and inflammatory diseases of the gastrointestinal tract. This research sheds important light into the pathogenesis of L. intracellularis and the host response associated with the lesions caused by infection. Lawsonia intracellularis causes porcine proliferative enteropathy. This is an enteric disease characterized by thickening of the wall of the ileum that leads to decreased growth of animals and diarrhea. In this study, we investigated the host response to L. intracellularis infection by performing transcriptomic and pathway analysis of intestinal tissue samples from groups of infected and noninfected animals at 14, 21, and 28 days postchallenge. At the peak of infection, when animals developed the most severe lesions, infected animals had higher levels of several gene transcripts involved in cellular proliferation and inflammation, including matrix metalloproteinase-7 (MMP7), transglutaminase-2 (TGM2), and oncostatin M (OSM). Histomorphology also revealed general features of intestinal inflammation. This study identified important pathways associated with the host response in developing and resolving lesions due to L. intracellularis infection. Lawsonia intracellularis is a Gram-negative, obligate intracellular bacterium that infects enterocytes of swine, primarily in the ileum, and causes a disease called proliferative enteropathy (PE)  . Infectethy (PE) . PE is ethy (PE) , 2, and  species .In swine, there are two major clinical forms of disease: proliferative hemorrhagic enteropathy (PHE) and porcine intestinal adenomatosis (PIA). PIA is a persistent but self-limiting disease that occurs in young pigs and can lead to diarrhea and reduced growth and is commonly a subclinical disease . PHE occL. intracellularis. This gap in knowledge is partly due to the fastidious nature of the organism, the difficulty growing pure cultures of the organism, and the lack of in vitro models that replicate proliferative lesions  and transforming growth factor β (TGF-β) have been found in the infected intestinal mucosa  had IHC and H&E stain scores of 1, indicating minor (low) lesions, and one of these animals had mild gross lesions. All three animals with H&E stain and IHC scores above 2 at 21 dpi had gross lesions, and one of these animals had severe gross lesions  and hematoxylin and eosin (H&E) microscopic lesion scores of one or zero with or without the presence of gross lesions in the group that received the challenge. “High” lesion animals were defined as those that had an IHC score of 2.5 and above, H&E score of 2 and above in addition to the presence of gross lesions. At 14 days postinfection (dpi), all infected animals had IHC and H&E stain scores of 1, indicating low-level infection with minor lesions, and no animals had gross lesions . At 21 d lesions . At 28 d lesions . None ofL. intracellularis bacteria at 21 dpi, when cycle threshold (TC) values were the lowest among all three time points . At 28 dpi, two animals in the infected group did not shed detectable levels of L. intracellularis in feces. In the noninfected group, two animals shed L. intracellularis . Since these animals did not have microscopic lesions measured using H&E stain or IHC and did not have positive antibody responses, they were still included in the study. Antibodies against L. intracellularis were not detected in serum samples of any of the animals in the noninfected group. Five of the six animals in the infected group had detectable antibody titers at 21 and 28 dpi. Antibody titers ranged from an average of 1:20 at 14 dpi to 1:920 at 28 dpi  serologic assay are shown in t 28 dpi .P < 0.05) (P < 0.05) (P < 0.05)  . At 21 d < 0.05) . While c < 0.05) .L. intracellularis infection. When we compared samples from challenged and nonchallenged pigs, no altered transcript abundance profiles were observed at 14 days postchallenge. However, at 21 dpi, there were 22 differentially expressed genes (DEGs), while at 28 dpi, only a single DEG was identified.To determine whether tissue lesions corresponded to differences in the abundance profile of gene transcripts expressed in the ileal samples, RNA-seq was employed to compare mRNA profiles between challenged and nonchallenged pigs and between pigs with high and low lesions due to XDH) and cytidine deaminase (CDA) (GABRP) was also among the genes with the greatest transcript abundance. Two matrix metalloproteinase (MMPs) gene transcripts were more abundant; these were MMP7 and MMP13.Among the 22 DEGs at 21 dpi, two were involved in nucleotide metabolism, xanthine dehydrogenase (se (CDA) . The genSAA1), whose inducible expression is a hallmark of the acute-phase response (OSM), a cytokine involved in diseases with chronic inflammation (SFRP5), an anti-inflammatory adipokine (TGM2), a functionally complex protein that is induced by inflammatory mediators . Other gene transcripts with greater abundance at 21 days postinfection included the tumor-associated calcium signal transducer gene (TACSTD2) and the gene transcripts that code for dual oxidase maturation factor 1 and 2 (DUOXA2 and DUOXA1).Genes described as being expressed by antigen-presenting cells, including macrophages and dendritic cells, were also differentially expressed. These included UNC5B). Its expression level was 3.77-fold higher in infected animals (P < 0.05) than in noninfected animals.The single gene transcript differentially expressed at 28 dpi encoded UNC-5 netrin receptor B (n = 494) observed than when comparing all infected animals together to noninfected animals (n = 22). At 28 dpi, the comparison between high- and low-lesion animals was different than that at 21 dpi, because two of the three low-lesion animals had either a negative H&E stain score or a negative IHC score and did not shed L. intracellularis at this time point, suggesting they had cleared infection or had a minimal level of infection. At 28 dpi, there were 23 DEGs when comparing animals with high lesions to those with low or no lesions. Because pigs within the challenged group at 21 dpi were classified by severity of infection (low versus high lesions), we wanted to know if host gene transcript expression was different between these two groups. Therefore, animals with high lesions were compared to those with low or minimal lesions. With this comparison, there were many more differentially expressed genes , bradykinin receptor B1 (BDKRB1), TNF-α-induced protein 6 (TNFAIP6), and integrin subunit alpha 8 (ITGA8). Among the most decreased gene transcripts in high- compared to low-lesion animals at 21 dpi were those of two genes that encode the apolipoproteins C-III (APOC3) and A-IV (APOA4) and the gene that encodes fibroblast growth factor 19 (FGF19). These code for proteins which are expressed in the small intestine and play important roles in lipid metabolism, among other functions . IDO1, which encodes indoleamine 2,3-dioxygenase, was increased in infected animals at 21 dpi, as well as in high-lesion animals at 28 dpi. The transcripts for the gene MGP, as well as those of genes TMEPAI and HTRA3, which have been found to play roles in apoptosis and influencing cell proliferation, were increased in high-lesion animals at both 21 and 28 dpi  with the DEGs, and the most significant was “cell proliferation of tumor cell lines” (P = 1.4E−15) (P < 0.05)  . Ingenui < 0.05) . Of the  < 0.05) .Next, we used IPA to identify canonical pathways associated with the DEGs between high- and low-lesion animals at 21 dpi. Of these pathways, we searched for pathways with a significant activation status. This analysis revealed 10 significantly activated canonical pathways associated with the data set (z-score of ≥2) .P = 2.06E−36), tumor protein p53 , and Erb-b2 receptor tyrosine kinase 2  were among the upstream regulators most significantly associated with the data set. Other significant upstream regulators included the cytokines transforming growth factor beta 1 , tumor necrosis factor , interleukin-1β (IL-1β), and IL-6 . We observed that the distribution of staining of this molecule differed between infected and noninfected animals. Staining of MMP-7 was not associated with hyperplastic crypts. MMP-7-positive cells were observed in normal crypts adjacent to those undergoing hyperplasia. Staining was observed both in the apical side of enterocytes and associated with the lumen of crypts . Infection was also confirmed by quantifying L. intracellularis-infected intestine tissue samples at different time points by microarray analysis and found that the number of DEGs coincided with the quantity of L. intracellularis bacteria present in tissue. This indicates that there is not a robust host response during early stages of the infection, and it coincides with lower lesions. At 21 dpi, when animals developed severe lesions, there were 22 DEGs between infected and noninfected animals. When comparing animals with high and low levels of infection and high and low lesions at this time point, there were 494 DEGs, again suggesting that the development of severe lesions is associated with a greater host response. Several of the genes found to be differentially expressed in this study were not observed by Smith et al. (P = 5.86E−23 to 1.9E−05). Differences in gene expression between both studies may be due to differences in lesion severity caused by infection and/ordifferences between microarray and RNA-seq methods of gene expression analysis.In this study, at 14 dpi, the lack of differentially expressed genes coincided with low lesion severity and antigen load. This was also observed by Smith et al. , who anah et al. , althougXDH   , which e= 0.009) , 23. Thillularis .MMP7, which encodes matrix metalloproteinase-7, or matrilysin, was also a transcript that was found to be upregulated in infected animals and in animals with high lesion scores at 21 and 28 dpi. This protein can be induced by some Gram-negative bacteria, and it has an important role in activating α-defensin antimicrobial peptides . This function had the involvement of 98 of the 494 DEGs among animals with high lesion scores at 21 dpi, which also correlated with the time when peak of crypt depth was noted using histomorphometry  and tumor protein p53 , are also associated with cell proliferation. CDKN1A inhibits the cyclin kinases that are necessary for cell cycle progression during the G1 and S phases and has an antiproliferative effector function. This gene is induced by p53 tumor suppressor protein, which is activated upon encounter with cellular stressors to induce the expression of genes that result in inhibition of proliferation  promotes cellular proliferation, and this gene is highly expressed in a wide variety of epithelial tumors (TGM2) can also promote cell proliferation, although this protein has many other functions , which had a 3.77-fold-higher abundance in infected animals (P < 0.05). This gene encodes a transmembrane receptor that can regulate cell fate as a “dependence receptor” and induces apoptosis in the absence of its ligand . Carbonic anhydrases can be found along the entire gastrointestinal tract, and in the small intestine, carbonic anhydrase 1 is found in cryptal enterocytes . Two groups of 18 animals were used in this study. One group was infected with me virus . AnimalsL. intracellularis infection   lesion was scored as either mild score of 1), moderate (score of 2) or severe (score of 3). For histopathology and evaluation of microscopic lesions characteristic of PE, a section of ileum proximal to the ileocecal valve was collected from each animal. This section of the intestine was chosen as it has been described as the most consistent site of , moderatnfection . The secrotocols . The preantigen) . The patTo assess differences in crypt and villus morphology due to infection, intestinal sections from animals were evaluated by measuring crypt depth and villus height. For this measurement, images were captured from slides using light microscopy with a 10× objective and analyzed with ImageJ . Twenty L. intracellularis in serum samples were measured using the immunoperoxidase monolayer assay (IPMA) (L. intracellularis was measured using qPCR (Antibodies against y (IPMA) . Fecal sing qPCR .Sus scrofa 3.0 (susScr3) genome was used, and gene annotation was performed using Ensembl from the same genome build. Differentially expressed gene (DEG) analysis was performed using CLC Genomics Workbench  and EdgeR. EdgeR has been described to have superior specificity and sensitivity, as well as good control of false-positive errors, compared to other methods to detect DEGs , and total RNA was extracted using the RNeasy plus universal minikit (Qiagen). RNA quantity and quality were assessed using the RiboGreen RNA assay kit and by capillary electrophoretic sizing using an Agilent TapeStation/Bioanalyze, respectively. Samples that passed quality metrics were used to create a cDNA library for sequencing using an Illumina TruSeq RNA Sample Preparation Kit. Samples from each pig were individually sequenced with the Illumina HiSeq 2500 sequencer using 50 paired-end reads; 20 million reads per sample were obtained. The Illumina sequence files were processed using a pipeline developed by the University of Minnesota Informatics Institute. Briefly, FastQ files were trimmed via trimmomatic and mapping was performed via TopHAT (version 2.0.13) using Bowtie (version 2.2.4.0). The ect DEGs . IngenuiComparisons were made between infected and noninfected animals at each time point postinfection, as well as between animals with high- (severe and diffuse) and low-level lesions. At 21 dpi, animals with high lesions (1381 and 97) were compared to the animals with low lesions  . RNA couMMP7, IHC was performed on paraffin-embedded tissue collected at necropsy. IHC of matrix metalloproteinase-7 was performed using a monoclonal antibody against MMP-7 following an adaptation of a previously described protocol , which is encoded by the gene protocol , using tGSE122764.The entire set of supporting gene expression and genomic data can be found in the Gene Expression Omnibus (GEO) database under accession number


Fifty-six percent of patients had a suboptimal vitamin C status even before surgery. In protein-denaturized probes, significantly higher vitamin C concentrations were detected (p = 0.0008). Vitamin E levels decreased significantly from preoperative level 11.6 (9.5–13.2) mg/L to 7.1 (5.5–7.4) mg/L, (p = 0.0002) at the end of cardiopulmonary bypass, remained low during the first day on ICU and recovered to 8.2 (7.1–9.3) mg/L 48 h after surgery. No patient was vitamin E deficient before surgery. Analysis showed no statistically significant association of vitamin C with inflammation, oxidative stress or organ dysfunction levels in patients with previously suboptimal vitamin C status or patients with a perioperative decrease of ≥50% vitamin C after surgery. Patients with higher vitamin C levels had a shorter ICU stay than those who were vitamin C depleted, which was not statistically significant . Conclusion: Vitamin C and E levels significantly declined intraoperatively and remained significantly reduced low for 2 days after cardiac surgery. The influence of reduced serum levels on the inflammatory reaction and clinical outcome of the patients remain unclear in this small observational study and need to be investigated further. Given vitamin C´s pleiotropic role in the human defense mechanisms, further trials are encouraged to evaluate the clinical significance of Vitamin C in cardiac surgery patients.Background: Oxidative stress contributes to organ dysfunction after cardiac surgery and still represents a major problem. Antioxidants, such as vitamins C and E might be organ protective. Methods: The primary objective of this prospective observational study was the description to evaluate the perioperative vitamin C and E levels in 56 patients undergoing cardiac surgery with the use of cardiopulmonary bypass. The association of vitamin C with inflammatory reaction, oxidative stress, organ dysfunctions, and clinical outcomes were evaluated in an explorative approach. Results: Vitamin C levels decreased significantly from 6.5 (3.5–11.5) mg/L before surgery to 2.8 (2.0–3.9) mg/L 48 h after surgery ( Cardiac surgery is associated with oxidative stress and systemic inflammation, which both contribute to postoperative organ dysfunctions . StimuliCurrent evidence suggests that the strong antioxidant and pleiotropic vitamin C can positively influence many organ systems, such as the neurologic, respiratory, renal, coagulation, and inflammatory and cardiocirculatory systems. The biochemical effects of vitamin C and the currently available preclinical and clinical evidence for the possible benefits of vitamin C administration in patients undergoing cardiac surgery was discussed in great detail elsewhere . BrieflyVitamin E is a group of fat-soluble compounds, of which α-tocopherol is the most common and biologically active form to meet human vitamin E requirements ,16, prevclinicaltrials.gov (NCT 02488876) and performed in adherence to the Declaration of Helsinki.This prospective, observational study at University Hospital RWTH Aachen, Aachen/Germany enrolled patients undergoing cardiac surgery from September 2018 until May 2019. Written informed consent was obtained from all enrolled patients prior to surgery. All patients were scheduled for elective conventional open-heart surgery with the use of aortic cross-clamping, cardioplegic myocardial arrest, and CPB. Exclusion criteria were emergency operations, pregnancy, lack of informed consent, and age less than 18 years. The study was approved by the institutional review board , registered at All patients received standard hospital meals during their entire hospital stay and were fasted for 6 h preoperatively. No patient received vitamin supplements during their hospital stay. All patients underwent catheterization, general anesthesia, and cardiac surgery with CPB according to local clinical standards. After surgery, all patients were transferred to a cardiac surgery intensive care unit (ICU) and were weaned from sedation and mechanical ventilation according to our department’s standards. Nutrition was provided in line with our feeding protocol, which is in agreement with current international guidelines on nutrition in the ICU.All blood samples for vitamin C and E measurements were drawn according to a standardized protocol prior to surgery, after opening of the aortic cross-clamp, at ICU-admission, and at 24 and 48 h after surgery.The blood samples were collected in ethylenediaminetetraacetic acid (EDTA) tubes for vitamin C measurements and in serum tubes for vitamin E measurements. All blood probes were light-protected with tinfoil and immediately pushed into ice. The probes were centrifuged (3000 rpm for 10 min) as soon as possible . The supernatant was transferred into an Eppendorf-tube and the probes were frozen at −80 °C under light protection. A planned interim analysis including all patients enrolled in 2018 revealed very low vitamin C levels. The method was afterwards modified based on the literature search as follows: protein was removed in the obtained serum after the first centrifugation using a detergent for protein denaturation  in a 1:1 ratio. This step is necessary to precipitate protein, to stabilize the ascorbate, and to remove interfering substances, and uses either an acid or an alcohol. This procedure is often combined with the use of a metal chelator such as EDTA to prevent accelerated ex-vivo ascorbate oxidation by divalent metal cations ,18. ProbAll probes were sent frozen and light-protected to an external laboratory  for the determination of vitamin levels through high performance liquid chromatography. In current international and national recommendations, a fasting plasma vitamin C concentration below 50 µmol/L  is an indicator of a suboptimal status with risk of insufficiency, while clinical symptoms such as fatigue might occur below 20 µmol/L ,24,25. TBlood samples were drawn in serum tubes and treated (centrifuged and frozen) as described above except that the probes were neither immediately light-protected nor pushed into ice. Oxidative stress was assessed using the oxidation–reduction potential (ORP) and antioxidant capacity (AC), measured with the RedoxSYS Diagnostic SystemTM . A low ORP being a sum of all oxidants and reductants in the blood indicates low oxidative stress, while a high AC is a measure of good antioxidant defense.Interleukin 6 (IL6) and interleukin 10 (IL10) were determined with commercially available ELISA assays  according to the manufacturer’s instructions.All other laboratory and clinical outcome parameters were collected as part of the clinical routine from chart review.The inflammatory reaction was characterized through the measurement of the inflammatory mediators C-reactive protein (CRP), procalcitonin (PCT) as well as white blood count (WBC). Their measurement was performed at indication of the attending medical staff as part of the clinical routine.Organ dysfunctions were assessed using the Sequential Organ Failure Assessment (SOFA) Score according to the original description . SOFA scWe compared patients, with a preoperative suboptimal vitamin C status to patients with normal vitamin C levels (cutoff: 9 mg/L), as well as patients who had a perioperative loss of vitamin C of ≥50% to those patients who did not lose vitamin C.p < 0.05 were considered statistically significant test results. The analysis was performed using SAS version 9.4 .Continuous variables were described by median and interquartile range (IQR) and categorical variables by counts and percentages. Group differences were tested by the Mann–Whitney U-test for continuous variables and the Fisher exact test for the categorical variables. Before–after comparisons were analyzed by paired Wilcoxon test. Due to the explorative study design, two-sided tests were used and Fifty-six patients were consecutively enrolled in this study . In a scPatient characteristics are described in p = 0.0002 (vitamin C) and 4.3 (3.4–5.5) mg/L, p = 0.0002 (vitamin E) corresponding to 66% (vitamin C) and 63% (vitamin E) of the respective baseline levels. Vitamin C levels remained significantly reduced until 48 h after surgery to , while blood levels of vitamin E remained reduced for 24 h and then recovered at 48 h  of their baseline levels as shown in Both vitamins decreased significantly during surgery with a median loss of 2.6 (0.7–5.3) mg/L, p = 0.0008) and 2.8 (2.0–3.9) mg/L 48 h after surgery (p = 0.0030)).Next, we investigated the influence of probe handling on the measured vitamin C levels in the blood. As shown in p = 0.6773), PCT levels (p = 0.4902), and SOFA Score (p = 0.1033). The CRP was measured in both groups of patients, when the attending medical staff indicated their measurement  levels were higher in patients with suboptimal preoperative vitamin C status at 96 and 120 h after surgery. PCT measurement as indicated by the attending medical staff was performed on 5 patients before surgery, 4 patients after surgery, 8 patients the first postoperative day, 9 patients at the second postoperative day, 7 patients at the third postoperative day, 6 patients at the fourth postoperative day, and 8 patients at the fifth postoperative day. Patients with a perioperative loss of vitamin C ≥ 50% compared to patients with perioperative loss < 50% did not show a difference with respect to the postoperative inflammatory response as assessed by mean postoperative WBC (p = 0.6625), CRP (p = 0.1318) and PCT levels (p = 0.5804), and SOFA Score (p = 0.1699), whereas the latter showed higher mean values of SOFA Score in patients with significant intraoperative vitamin C loss during the postoperative course, which however did not reach statistical significance  and IL10 (p = 0.7261) as shown in No significant differences between the groups with suboptimal preoperative vitamin C status versus preoperative normal plasma serum levels of vitamin C were observed regarding the perioperative course of interleukins, as assessed in 10 patients by mean IL6 (p = 0.6720) and AC (p = 0.4859) in 16 patients. Patients with a suboptimal preoperative vitamin C status showed visually higher ORP levels at the end of CPB and at ICU admission, which did not reach statistical significance in this small group of patients  and demonstrated a strong concordance between changes in plasma vitamin C concentration and changes in severity of oxidative stress, reflecting that a high dose of vitamin C diminished the oxidative stress in their critically ill patients [Our results regarding the oxidative stress are in line with the recently published study by Rozemeijer et al., who also observed an increase of oxidative stress in ICU-patients undergoing major surgery/sepsis or trauma, as well as in patients after cardiac arrest to similar levels we observed in patients undergoing cardiac surgery. Additionally, Rozemeijer et al. observed a strong negative association of ORP and strong positive association between the anti-oxidative capacity with plasma vitamin C concentrations —both in patients with higher preoperative levels, as well as in patients with smaller intraoperative vitamin C loss. Although these results did not reach statistical significance, this might be of clinical relevance, especially for patients with prolonged ICU-stay , and nee h—both iOur study has several limitations. The sample size was too small to allow for any conclusions regarding the association of the vitamin levels with the clinical outcome. Considering the measurement of IL6, IL10, and ORP, we must acknowledge that the present data should be interpreted cautiously within the limitations of an explorative analysis, which should be considered hypothesis-generating due to the low number of observations. Secondly, we included all patients consecutively without risk-stratification, therefore, recording low frequencies of adverse events, such as organ dysfunction or mortality in elective patients. Thirdly, we observed a high inter-individual variability of vitamin C, leading to the need of studies with greater patient numbers to draw meaningful conclusions. Fourthly, intraoperative treatments, such as the administration of blood products and colloids may have influenced the patient´s native vitamin C and E levels. Furthermore, as our laboratory values other than vitamins, oxidative status, and interleukins were taken as part of clinical routine, and all collected blood samples were used for these purposes, it was not possible for us to correct the vitamin E levels with blood lipids, or to perform additional measurements, for example, for other inflammatory markers. Yet, present finding of a significant decrease of these key micronutrients demonstrates the need for a more carefully clinical evaluation, especially in the post-acute phase of cardiac surgery patients.In conclusion, while the vitamin C and E plasma levels decrease dramatically during elective cardiac surgery with cardiopulmonary bypass and remain low in the first two postoperative days, their influence on clinical outcome remains unclear in this small observational study. The clinical relevance of vitamin C and E deficiency in patients undergoing cardiac surgery with cardiopulmonary bypass requires further investigation.