Neurotensin-Binding Immunoglobulin G in Patients with Parkinson’s Disease

Introduction: Neurotensin (NTS) is a 13-amino acid neuropeptide functionally linked with the brain dopaminergic system via expression of the NTS peptide or its receptor in dopamine neurons. Neuropeptide-binding immunoglobulins (Igs) are present in humans and can be involved in both physiological and pathological processes. Considering the functional link between NTS and dopamine neurons, we studied the occurrence of NTS-binding IgG autoantibodies in patients with Parkinson’s disease (PD). Methods: Plasma levels of NTS-binding IgG were analyzed using enzyme-linked immunosorbent assay in both male and female PD patents and in age-matched healthy controls. Possible microbial origin of NTS cross-reactive IgG was analyzed by sequence alignment of the 6-amino acid C-terminal NTS pharmacophore with bacterial and viral proteins from the public NCBI database. Results: NTS-binding IgG were detected in the plasma of all study subjects, while their levels were consistently lower in PD patients versus controls (p = 0.0001), independently from age or sex of the study participants. Moreover, PD patients with a more severe stage (2.5–3.0) of the disease had lower levels of NTS-binding IgG (p = 0.0004) than those with a milder stage (1.0–2.0). Furthermore, PD patients taking amantadine or high doses of levodopa had higher levels of NTS-binding IgG than those without medication. Contiguous sequence homology for the NTS pharmacophore was present in several microbial proteins occurring in human gut microbiota. Discussion: The study revealed that NTS-binding IgG occur naturally in humans and that PD patients display their low plasma levels accentuated by disease severity. The functional significance of this finding and its relevance to the pathophysiology of PD, including putative link to gut microbiota, remain to be studied.


Introduction
Neurotensin (NTS) is a 13-amino acid neuropeptide produced in the gut and the brain [1].Several studies showed functional and anatomical association between NTS and the dopamine (DA) system, with NTS displaying mainly a stimulatory effect on DA release [2][3][4].In particular, it has been shown that the NTS peptide is expressed in DA neurons of the hypothalamic arcuate nucleus and in a subset of neurons in the ventral tegmental area.In contrast, DA neurons of the substantia nigra (SN) do not contain NTS, but abundantly express NTS receptor (NTSR) in both humans and rodents [5,6].
The functional connections between NTS and the DA systems suggest that altered NTS signaling may be present and/or contribute to Parkinson's disease (PD).As such, postmortem studies in humans showed that the NTS binding density was decreased in the SN and other brain areas of subjects who had PD [5,7].Importantly, NTS along with several other neuropeptides display neuroprotective effects for the DA system and PD [8].Indeed, experimental studies in rodents showed that NTS analogs may protect against PD-like symptomatology [9,10].Moreover, NTSR can be used for the targeted delivery of neuroprotective factors' genes into the SN DA neurons [11].Taken together, NTS signaling appears to be relevant to the PD pathophysiology, although its exact contribution remains to be clarified.
There is accumulating evidence that several neuropeptides and peptide hormones circulate bound to immunoglobulins (Igs), which play a role of a peptide carrier protein modulating peptide biological activity.For instance, IgG binding α-melanocyte-stimulating hormone (α-MSH), a 13-amino acid anorexigenic peptide, have been implicated in the modulation of α-MSH signaling upon melanocortin receptor, a molecular mechanism relevant to eating disorders [12].Moreover, IgG binding to ghrelin, a hunger hormone, were shown to protect it from degradation in circulation and to enhance its orexigenic effects in obesity [13].The cortisol-stimulating effect of adrenocorticotropin can also be modulated by adrenocorticotropin-binding IgG, resulting either in enhancement or inhibition of cortisol secretion [14].However, the occurrence of NTS-binding IgG has not been reported.
The origin of neuropeptide-binding IgG may be related to molecular mimicry with homologous microbial antigens [15].In fact, sequence homology between microbial proteins and several peptide hormones have been noticed [16].Molecular mimicry between α-MSH and Enterobacteriaceae caseinolytic protease B (ClpB) underlies production of α-MSH cross-reactive IgG autoantibodies (autoAbs) [17].Whether the NTS peptide may display sequence homology with microbial proteins is presently unknown.
Thus, in the present study, we aimed at analyzing the occurrence of NTS-binding IgG autoAbs in humans and to compare their plasma levels between PD patients and healthy controls.We also studied if the levels of NTS au-toAbs may correlate with PD severity and medication as well as with cognitive function, depression, and anxiety scores.To address a putative antigenic origin of NTSbinding IgG in microbial proteins, we performed in silico search of sequence homology between both the full NTS peptide or its 6-amino acid C-terminal pharmacophore and bacterial, viral, and fungal proteins from the public National Center for Biotechnology Information (NCBI) database.

Participants
All procedures were in accordance with the Declaration of Helsinki and were approved by the Regional Committee for Medical and Health Research Ethics at the Institute of Experimental Medicine, Saint-Petersburg, Russia (protocol N5/20, July 08, 2020).All patients signed written informed consent prior to participation in the study.The study recruited 48 PD patients who were admitted at the IEM clinic between March 2021 and Dec 2021.Patients with vision and hearing impairment, premorbid clinically relevant psychiatric disorders, alcohol and narcotic addictions, inflammatory diseases, cancer, chronic diseases, and a history of major surgery were excluded.PD was diagnosed according to the Queen Square Brain Bank criteria (QSBB) [18] and confirmed by at least two movement disorder specialists.The stage of the disease was determined by the Hoehn and Yahr scale [19].Cognitive function was assessed using the Mini-Mental State Examination (MMSE) scale (scores 0-30) [20] and the Montreal Cognitive Assessment (MoCA) scale (scores 0-30).Affective symptoms were measured by the Beck Depression Inventory (BDI) scale (scores 0-63) [21], the Hospital Anxiety and Depression Scale (HADS, scores 0-21) [22], and the Hamilton Depression Rating Scale (HDRS, scores 0-52) [23].Majority of PD patients received anti-PD medication consisting of different doses of levodopa (L-DOPA) and/or amantadine (for the number of treated patients, see sections L-DOPA Treatment and Amantadine Treatment).Patients received medications according to a personal scheme.The study considered the daily dose of medications.A control group consisted of 48 healthy volunteers, who all underwent a medical examination (Table 1).All control group members exhibited no evidence of neurological or psychiatric disease or evidence of chronic or acute inflammatory disease, cancer, history of major surgery, vision and hearing impairments.Cognitive status and anxiety and depression scores were not analyzed in the control group.

Pre-Analytic Analysis and Sample Collection
Anti-parkinsonian drugs were stopped 16 h prior to blood sampling.Clinical nursing staff collected 5 mL of blood from the cubital vein of all patients and the control group volunteers in the morning following a night of fasting.Blood was allowed to clot for 30 min at room temperature and then centrifuged at 150-200 g for 10 min.The plasma was collected and aliquoted into fresh polypropylene vials and stored frozen at −80°C prior to NTS IgG assay.

NTS-Binding IgG Assay
The NTS-binding IgG levels were measured using an enzymelinked immunosorbent assay (ELISA) according to a published generic protocol for neuropeptide autoAbs assay [24].Briefly, synthetic human NTS peptide (QLYENKPRRPYIL) with 95% chemical purity (NPF Verta, St Petersburg, Russia) was coated on Costar 96-well microplates (Sigma, St. Louis, MO, USA) at 2 μg/mL in 100 mmol/mL NaHCO 3 buffer, pH 9.6, for 3 h at 37°C, while uncoated wells served for the blank values.Plasma samples were diluted 1:50 in phosphate-buffered saline (PBS), pH 7.4, or in sample dissociating buffer: 3 M NaCl, 1.5 M glycine, pH 8.9, and incubated overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated rabbit anti-human IgG (Sigma), 1:4.000 for 3 h at room temperature.Dilution of plasma with normal (PBS) or dissociating buffers allowed to estimate the levels of "free" or "total" NTS-binding IgG, respectively (Fig. 1).The optimal dilution was selected by serial dilutions of several plasma samples, showing linear detection levels for both free and total NTS-binding IgG (data not shown).To reduce the influence of inter-plate assays, equal number of samples from both control and PD groups were deposited on each ELISA plate.Washes in 0.05% Tween-20 in PBS were performed between all steps.Chromogenic reaction was induced by adding 3,3,5,5-tetramethylbenzidine solution (Sigma) for 40 min, and its optical density (OD) was read by an ELISA plate reader at 450 nm.The mean blank OD values were subtracted from the mean OD values obtained from NTS-coated wells measured in duplicate for each plasma sample and showing variance less than 5%.The blank OD values in different plates varied between 0.08 and 0.083.

Plasma Absorption with NTS
To verify the specificity of NTS-binding reactivity of plasma IgG in our ELISA test, we performed plasma absorption experiment.For this purpose, 3 plasma samples were randomly selected from both control and PD groups and were preincubated overnight at 4°C with 3 (×10) dilutions of NTS peptide (10 −8 , 10 −7 , and 10 −6 M) in PBS and then applied to the ELISA plate coated with NTS (Fig. 1).The NTS-binding IgG levels were measured as described in the previous section.The same plasma samples without preincubation with NTS peptide served as positive controls.

In silico Analysis of Sequence Homology between NTS and Microbial Proteins
Amino acid sequence homology between human full NTS peptide (QLYENKPRRPYIL) or its C-terminal pharmacophore (RRPYIL) and proteins from bacteria, fungi, and viruses were searched in public protein databases at the NCBI (Bethesda, MD, USA; www.ncbi.nlm.nih.gov) using the Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi).Global alignment of the sequences was performed using multiple sequence alignment with hierarchical clustering (Corpet, 1988).

Statistical Analysis
Descriptive statistics are presented as absolute numbers, percentages and median, and interquartile range.Plasma anti-NTS IgG was expressed as mean ± SEM.Student's t test was used for comparison between two groups.Correlations were analyzed using Spearman's rank correlation coefficient and Pearson's correlation coefficient between levels of total IgG and levels of free IgG in both PD and control groups.Correlation between disease severity, non-motor functions, and plasma anti-NTS IgG were analyzed using Kendall rank τ correlation coefficient (R tau ) and correlations between total and free IgG using the Spearman's and Pearson's tests.Fisher's exact test was used to determine if there were sex differences between the groups.A p value <0.05 was considered as statistically significant.Statistical analysis was performed using STATISTICA 8.0 (StatSoft, Tulsa, OK, USA).

Demographic and Clinical Characteristics of Study Subjects
The PD group of 48 patients had an average age of 68 years with an age range between 44 and 87 years, which was not significantly different from the control group of 48 volunteers (mean age 68 years with an age range 43-86 years).There was also no significant sex difference between the groups (Fisher's exact test, p = 0.86).For more demographic, clinical, and biochemical data, see Table 1.

NTS-Binding IgG Are Present in Human Plasma and Decreased in PD Patients
NTS-binding IgG were readily detected in the plasma samples from both healthy controls and PD patients.As compared to control subjects, PD patients had significantly lower plasma levels of both NTS-binding total and free IgG (Fig. 2a,b, t 4.63 and 5.83, respectively, p = 0.0001 for both).The levels of total and free IgGs strongly correlated between each other in both control and PD patients' groups (Spearman's r = 0.97 and Pearson's r = 0.98, respectively, p < 0.0001 for both, Fig. 1d).Nevertheless, the ratios of total to free IgG levels were significantly lower in PD patients (t = 5.53, p = 0.0001, Fig. 2c).Absorption of plasma with the synthetic NTS peptide prior to the detection of NTS-binding IgG resulted in a dose-dependent decrease of its detection levels in all samples from both controls and PD patients.Such a decrease was observed after preincubation of plasma with 10 −8 M NTS peptide, and it was more pronounced with higher NTS concentrations (Fig. 2e).There was no significant relationship found between NTS IgG levels and age either in PD (R = −0.09,p > 0.05) or in control groups (R = −0.11,p > 0.05) and with sex, PD group (t = −0.03,p = 0.97), control group (t = 0.05, p = 0.94).No significant associations of NTS-binding IgG levels with the presence of constipation in PD patients were observed (data not shown).

NTS-Binding IgG Correlate with PD Severity
All PD patients were divided into two subgroups depending on the disease stage as reflected by the severity of motor dysfunction, according to the Hoehn and Yahr scale.The first subgroup included patients with stages 1.0-2.0(subgroup PD1, n = 25, 10 males and 15 females), the second, with stages 2.5-3.0 (subgroup PD2, n = 23, 10 males and 13 females).Plasma levels of NTS-binding IgG were compared between the subgroups.The subgroup PD2 had significantly lower plasma levels of both total and free NTS-binding IgG (t = 2.53, p = 0.015 and t = 2.40, p = 0.02, respectively) as compared to the PD1 (Fig. 3a,c, respectively).We observed weak negative correlation between the plasma total level of anti-NTS IgG and the disease stage (R tau = −0.33,p < 0.05, Fig. 3b), as well as between the plasma-free level of anti-NTS IgGs and the disease stage (R tau = −0.31,p < 0.05, Fig. 3d).There was no relationship found between the ratio of total IgGs to free IgGs and disease stage (Fig. 3f).

NTS-Binding IgG Levels Are Associated with anti-PD Medication L-DOPA Treatment
The PD group was divided into three subgroups depending on the dose of L-DOPA medication: (1) subgroup without L-DOPA treatment PD L-dopa0 (n = 16); (2) subgroup with low doses (187.5-600mg/day) PD L-dopa L (n = 15), and (3) subgroup with high doses (600 mg/day to 1,200 mg/day) PD L-dopa H (n = 17).We found that the use and dose of L-DOPA affects plasma levels of both total (F(2, 45) = 13.1, p = 0.0003) and free (F(2, 45) = 14.86, p = 0.0001) NTS-binding IgG.No significant link between L-DOPA and the ratio of total IgG to free IgG was found (F(2, 45) = 1.7, p = 0.19).When conducting a multiple comparison, it turned out that in all patients receiving L-DOPA at a dose of 600 mg/day and in patients receiving the drug at a dose exceeding 600 mg/day, the plasma levels of NTS-binding total IgG were higher than in patients not receiving any L-DOPA therapy or receiving its lower doses (PD L-dopa0 vs. PD L-dopa H , p = 0.0001 and PD L-dopa L vs. PD L-dopa H , p = 0.018, respectively, Fig. 6a).The plasma levels of NTS-binding free IgG were also significantly increased in subgroups receiving higher L-DO- PA dosage (PD L-dopa0 vs. PD L-dopa H , p = 0.0001, PD L-dopa0 vs. PD L-dopa L , p = 0.04, and PD L-dopa L vs. PD L-dopa H , p = 0.02, respectively, Fig. 6b).

NTS Displays Molecular Mimicry with Microbial Proteins
Protein BLAST search in bacteria, viruses, and fungi was conducted in two conditions: (1) with the full 13-amino acid sequence of the human NTS peptide and ( 2) with only the 6-amino acid pharmacophore sequence of the NTS C-terminal.Among the obtained results, only those were selected which had the input sequence fully covered, i.e., for which the identity was 100% and for which the source microorganisms have been found in human microbiota.The BLAST search revealed that there were no proteins with 100% identity to the full NTS sequence in bacteria, fungi, or viruses.In contrast, several bacterial and viral proteins were found containing fragments displaying 100% homology to the last 6 amino acids of hu- man NTS, as shown in Table 2.No such homology was detected in fungal proteins.We would like to remind that potential NTS-like activity of these in silico-identified bacterial proteins should be validated by in vitro and in vivo experiments.

Discussion
The main results of this study were the following: (1) NTS-binding IgG are present naturally in humans; (2) plasma levels of NTS-binding IgG are lower in PD patients than in healthy subjects, and (3) amino acid sequence identity exists between the NTS C-terminal pharmacophore and several bacterial and viral proteins.Below, we discuss possible functional significance of these findings.We admit that the descriptive nature of this study will limit the discussion to speculations about such functional significance based on existing literature data.
The finding of the natural presence of NTS-binding IgG in healthy humans is not surprising.Although such autoAbs have not yet been reported, these results add to the list of previously detected neuropeptide-binding IgG autoAbs, which may reflect a general phenomenon implicating IgG in peptidergic signaling and functionally linking gut microbiota and the brain [16,25].In fact, recent data show functional implication of IgG as natural carrier proteins of peptide hormone which preserve and modulate their biological activity [12][13][14].Thus, the putative functional role of NTS-binding IgG may also consist of preservation of NTS signaling.In fact, previous studies analyzing NTS plasma concentrations reported that the NTS peptide was bound to plasma proteins, but the nature of such proteins was unknown [26].In the present study, we found that levels of NTS-binding free and total IgG were similar, although not identical in the same subjects, underlying their strong correlations in the study group (Fig. 1d).This is an unusual finding for a neuropeptide-binding IgG which are typically detected at higher levels of total versus free IgG, reflecting high levels of such IgG present as immune complexes, e.g., hypocretin-binding IgG [27].Thus, according to our results, it appears that NTS-binding IgG do not form stable IC.
The present study also revealed possible relevance of NTS-binding IgG to PD by showing their lower plasma levels in PD patients.Moreover, a relative binding capacity of IgG to NTS was compared between PD patients and controls by calculating the ratios of total to free NT-binding IgG levels.A significant decrease of such ratios was observed in the PD group and it was not related to the disease severity and PD medication.Such a decrease may signify lower NTS-binding capacity of IgG in PD patients.The absorption experiment showed a specificity of NTS binding by plasmatic IgG and indicated that such interactions may occur with a 10 −8 M affinity, which is typical for neuropeptide-reactive IgG.Future analysis of IgG affinity kinetics for NTS should clarify better these findings.Thus, a significant decrease of both NTS-binding IgG levels and their binding capacity in PD patients may be interpreted as a decrease of their NTS-carrier function resulting in lower NTS signaling.Previous study showed lower NTS immunoreactivity in crude extracts of postmortem brain tissue of PD patients, while after HPLC purification, resulting in a 3-fold decrease of NTS detection, its levels were higher in the SN of PD patients versus controls, suggesting an activation of local NTS innervation of the SN induced by loss of DA [28].Moreover, Schimpff et al. [26] reported elevated plasma levels of NTS in PD patients, although the majority of NTS-like immunoreactivity was detected in protein fractions with higher than NTS molecular weight.We may speculate that some NTS-like positive material, which is not NTS or its precursor, could be represented by naturally present microbial proteins displaying molecular mimicry to NTS and derived from the human microbiota.Below, we discuss a possibility that such proteins may play a role of natural antigens underlying the presence of NTS-binding IgG.
NTS is produced in the brain but also in the enteroendocrine cells of the gut [29,30].Peripheral administration of NTS analogs in a rat model of PD reduced the symptoms severity, while in vitro studies show its potent neuroprotective effect [9,10,31].Whether peripheral NTS bound to IgG may reach the brain and protect DA neurons is presently unknown.It is likely that peripheral IgG may slowly pass across the blood-brain-barrier resulting in functional interstitial IgG levels, as has been shown for both human and rat IgG using microdialysis in the rat brain [32].It is, hence, possible that low levels of NTSbinding IgG found in PD patients may contribute to the PD pathophysiology via deficient NTS transport and signaling.In this context, it is noticeable that PD patients with a more severe motor dysfunction displayed even lower levels of NTS-binding IgG.Moreover, use of anti-PD medication, both L-DOPA and amantadine, were associated with higher levels of NTS-binding IgG, and higher doses of L-DOPA were linked to their even higher levels.While of relevance to PD symptomatology, it is difficult to speculate about possible causal relations of this finding, which may also reflect an indirect effect of medication on the immune system or gut microbiota.It should Neuroimmunomodulation 2023;30:15-27 DOI: 10.1159/000527872 be noticed that higher levels of plasma NTS were found in L-DOPA-treated versus untreated patients [26]; these data favor a role of NTS-biding IgG in increasing NTS stability.
Besides its putative central action, IgG-mediated altered NTS signaling may contribute to several peripheral effects, e.g., gastrointestinal manifestations typically occurring in PD patients [33].In fact, NTS is a well-known modulator of gut motility and can also be involved in colonic inflammation [34,35].Moreover, NTS was shown to inhibit corticotropin-induced cortisol secretion in the adrenal gland, i.e., it may contribute to the regulation of stress response and glucose metabolism [36].
Since NTS may modulate DA and other neurotransmitter release and signaling, i.e., relevant to both motor and non-motor functions [37], we studied if NTS-binding IgG levels may correlate with scores obtained from 4 different scales evaluating memory, cognition, mood, and emotion.The scores reflecting the full spectrum of behavior were obtained in each scale from all study subjects.However, none of the scales had significant correlations with the levels of NT-binding IgG.These results point to rather a selective link between NT-binding IgG and the altered motor function characteristic for PD, which may be related to a high expression of NTSR in SN neurons [5].Additionally, NTS was shown to modulate neuroinflammation via activation of microglia [38].
Several studies explored the putative mechanistic link between gut microbiota and PD, and were recently reviewed [39][40][41].Nevertheless, in spite of some differences in gut microbiota composition found in PD patients, there is so far no clear pathophysiological model implicating gut bacteria in the origin and/or maintenance of PD.Our working hypothesis proposes that neuropeptide-binding autoAbs, which can be necessary for the normal neuroendocrine signaling by neuropeptides, are produced mainly in response to antigenic stimulation by microbial proteins from gut microbiota due to their molecular mimicry with neuropeptides.This hypothesis was previously validated for the origin of α-MSH IgG autoAbs [17].In the present study, to approach the possible microbial origin of NTS-binding IgG, we searched for the sequence homology between NTS and microbial proteins.Several such proteins with fragments identical to the 6-amino acid C-terminal of the NTS pharmacophore were found belonging to specific bacteria.Their possible relevance to PD is discussed below.We should also mention that the NTS-pharmacophore shares 4 amino acids with the C-terminal of neuromedin N, a 6-amino acid neuropeptide derived from the same precursor as NTS and having similar with NTS functions [42].
Decreased levels of NTS-binding IgG in PD patients may signify lower antigenic load by NTS-cross-reactive antigens.The meta-analysis of 15 human studies exploring gut microbiota composition in PD revealed that several bacterial families were significantly increased or underrepresented [43].The latter group was mainly represented by Fecalibacterium of the Oscillospiraceae family and by the Lachnospiraceae family, both belonging to short-chain fatty acids-producing class of Clostridia of the phylum of Firmicutes [44,45].Thus, considering the consistent data of lower levels of these bacteria in gut microbiota of PD patients, our finding of sequence homology between NTS and the MFS protein from both Clostridiales and Oscillospiraceae bacteria suggests that low production of such bacterial protein may contribute to low levels of NTS cross-reactive IgG in PD.With regard to the presence of NTS sequence homology in Eggerthella species, an increased abundance of these bacteria was reported in gut microbiota of monkeys with experimental PD [46].Of interest, an enzyme produced by Eggerthella lenta was shown to reduce efficacy of L-DOPA medication by converting DA into tyramine [47].We may speculate that L-DOPA could promote the growth of its metabolizing bacteria which may eventually underlie increased NTS-like IgG reactivity in PD patients under such medication.
Another NTS sequence homology was detected in a protein from Sanguibacteroides justesenii.This bacterium belongs to the Porphyromonadaceae family.Its increased presence in gut microbiota was earlier associated with impaired cognitive function in patients with neurodegenerative diseases including PD; however, the relevance of this particular species to human health is presently unknown [48].Discussion on the possible link of NTS sequence homology with a protein from Siphoviridae is limited to the data from one study reporting a slightly lower abundance of this bacteriophage family in the gut microbiota of PD patients [49].Future experimental immunization studies should determine if any of the microbial proteins identified by our in silico analysis are able to stimulate production of NTS cross-reactive IgG and if such IgG may enhance neuroprotective effects of NTS in animal models of PD.A putative direct effect of NTS-like microbial proteins on DA release is another interesting possibility in the mechanisms of microbiota-brain interactions in the context of PD.
In conclusion, our study demonstrated for the first time the natural occurrence of NTS-binding IgG in hu-

Fig. 1 .
Fig. 1.Schematic drawing of the "home-made" ELISA technique used for the detection of NTS-binding IgG.For details, see the methods section.

Fig. 2 .Fig. 3 .Fig. 4 .Fig. 5 .
Fig. 2. NTS-binding IgG in PD and control groups.a Plasma total NTS IgG.b Plasma-free NTS IgG.c Ratio of total IgG to free IgG.Data are shown as means ± SEM (n = 48 for each group), t test.d Correlation between levels of total IgG and free NTS-binding IgG in PD and control groups (n = 96).e Effects of absorption of 3 controls' (C1-3) and 3 PD patients' (PD1-3) plasma samples with different concentrations of NTS peptide on detection of NTS-binding IgG.

Fig. 6 .
Fig. 6.The impact of L-DOPA therapy in PD patients on their plasma levels of NTS-binding total IgG (a), free IgG (b), and their ratios (c).Data are expressed as means ± SEM, the volume of each group is indicated in the text, one-way ANOVA, Tukey post hoc tests, *p < 0.05 and ***p < 0.001.

Fig. 7 .
Fig. 7.The impact of amantadine therapy in PD patients on their plasma levels of NTS-binding total IgG (a), free IgG (b), and their ratios (c).Data are expressed as means ± SEM, the volume of each group is indicated in the text, one-way ANOVA, Tukey post hoc tests, **p < 0.01 and ***p < 0.001.

Table 2 .
Bacterial and viral proteins with 100% identity to the biologically active part of human NTS (RRPYIL) DOI: 10.1159/000527872