Blood profile holds clues to role of infection in a premonitory state for idiopathic parkinsonism and of gastrointestinal infection in established disease

The two-stage neuroinflammatory process, containment and progression, proposed to underlie neurodegeneration may predicate on systemic inflammation arising from the gastrointestinal tract. Helicobacter infection has been described as one switch in the pathogenic-circuitry of idiopathic parkinsonism (IP): eradication modifies disease progression and marked deterioration accompanies eradication-failure. Moreover, serum Helicobacter-antibody-profile predicts presence, severity and progression of IP. Slow gastrointestinal-transit precedes IP-diagnosis and becomes increasingly-apparent after, predisposing to small-intestinal bacterial-overgrowth (SIBO). Although IP is well-described as a systemic illness with a long prodrome, there has been no comprehensive overview of the blood profile. Here, it is examined in relation to Helicobacter status and lactulose-hydrogen-breath-testing for SIBO. A robust finding of reduced lymphocyte count in 126 IP-probands and 79 spouses (without clinically-definite IP), compared with that in 381 controls (p < 0.001 in each case), was not explained by Helicobacter-status or breath-hydrogen. This complements a previous report that spouses were 'down-the-pathway' to 'clinically-definite' disease. In 205 other controls without clinically-definite IP, there were strong associations between sporadic cardinal features and immunoglobulin class concentration, not explained by Helicobacter-status. Premonitory states for idiopathic parkinsonism associated with relative lymphopenia, higher serum immunoglobulin concentrations and evidence of enteric-nervous-system damage may prove viral in origin. Although only 8% of the above 79 spouses were urea-breath-test-positive for Helicobacter, all 8 spouses with clinically-definite IP were (p < 0.0001). Transmission of a 'primer' to a Helicobacter-colonised recipient might result in progression to the diagnostic threshold. Twenty-five percent of the 126 probands were seropositive for anti-nuclear autoantibody. In 20 probands, monitored before and serially after anti-Helicobacter therapy, seropositivity marked a severe hypokinetic response (p = 0.03). It may alert to continuing infection, even at low-density. Hyperhomocysteinemia is a risk factor for dementia and depression. Serum homocysteine exceeded the target in 43% of the 126 IP-probands. It was partially explained by serum B12 (12% variance, p < 0.001), but not by Helicobacter-status (gastric-atrophy uncommon in IP) or levodopa treatment. Immune-inflammatory activation increases homocysteine production. Since an estimated 60% of probands are hydrogen-breath-test positive, SIBO, with its increased bacterial utilisation of B12, is a likely cause. Thus, two prognostic indicators in established IP fit with involvement of Helicobacter and SIBO.

Although only 8% of the above 79 spouses were urea-breath-test-positive for Helicobacter, all 8 spouses with clinically-definite IP were (p < 0.0001). Transmission of a 'primer' to a Helicobactercolonised recipient might result in progression to the diagnostic threshold.
Twenty-five percent of the 126 probands were seropositive for anti-nuclear autoantibody. In 20 probands, monitored before and serially after anti-Helicobacter therapy, seropositivity marked a severe hypokinetic response (p = 0.03). It may alert to continuing infection, even at low-density. Hyperhomocysteinemia is a risk factor for dementia and depression. Serum homocysteine exceeded the target in 43% of the 126 IP-probands. It was partially explained by serum B12 (12% variance, p < 0.001), but not by Helicobacter-status (gastric-atrophy uncommon in IP) or levodopa treatment. Immune-inflammatory activation increases homocysteine production. Since an estimated 60% of probands are hydrogen-breath-test positive, SIBO, with its increased bacterial utilisation of B12, is a likely cause. Thus, two prognostic indicators in established IP fit with involvement of Helicobacter and SIBO.

Background
It has been proposed [1] that, whatever the aetiological insult or affected brain area, a two-phase neuroinflammatory process, containment and progression, is common to neurodegenerative diseases. These two phases may predicate on systemic inflammation.
Confirming relative lymphopenia in probands would indicate a xenobiotic, nutritional or infective influence, finding it in their spouses a shared insult in adult-life. In IP, there is no convincing evidence of excessive exposure to xenobiotics, or of the relevance of genes regulating their metabolism [18]. There is no indication of malnutrition/ malabsorption, but increased energy expenditure may cause weight loss [24]. Regarding possible haematinic deficiency, peptic ulcer is prodromal [26]. Lymphopenia might result directly from chronic infection. H. pylori whole-bacteria, cell-fractions, culture-supernatants and protein-products, and H. pylori-specific regulatory T-lymphocytes inhibit human T-cell proliferation [27,28]. Moreover, a lower serum IgM is associated with Helicobacter-seropositivity [29], but whether the relationship holds in IP is unknown. Lymphopenia could result from autoimmunity, reversible by eradicating a trigger, as proposed in Helicobacter-associated idiopathic thrombocytopenic purpura (ITP) [30]. Indeed, antibody against a H. pylori virulence-factor, cytotoxicity-associated-gene (cagA) product, is implicated in ITP [30] and IP [22]. In the presence of SIBO, most IP-probands' duodenal enterocytes contain apparently hypertrophic mitochondria [18]: lymphocyte mitochondria might show similar morphological compensation for hypofunction [31].
We present the first comprehensive overview of the blood profile in IP in relation to indices of Helicobacter infection and hydrogen-breath-testing for SIBO. We consider definition of a 'premonitory' stage, potential markers of progression towards established disease and prognostic indicators within it. An aetiological/pathogenic solution may remain elusive without embracing limited manifestations in 'controls'.

Patients and controls
In phase-1 [22], we recruited consecutive patients with 'clinically-definite' IP [32], criteria as in Table 1. There was a contemporaneous call for healthy controls without IP: those found to have 'clinically-possible or -probable' parkinsonism [32] (Table 1, footnote a ) were not excluded. Other criteria were as shown for probands in the Table. Serum immunoglobulin concentrations were contrasted between 120 probands (12 men, 12 women per decade, 40-89 years) and 205 controls (≥12 each gender per decade, 30-89 years). The relationship of presence/absence of features of clinically-possible or -probable IP to concentration was explored in controls, as was that of a continuous measure of hypokinesia, mean-stride-length at freewaking-speed [33]. Any differential effect of Helicobacter anti-urease antibody-status was examined.
In phase-2 [23], we obtained first-visit full blood counts (FBC) from 118 other clinically-definite IP-probands (Table 1). These were contrasted with 381 routine FBCs from consecutive adults, requested by general practices from the same laboratory over the same period. Exclusions in these controls were limited to specified parkinsonism; haematological, immunological and malignant disorders; haemorrhage; and race other than Caucasian (see footnote b ). FBCs from 87 accompanying spouses/ partners of probands were also studied, inclusive of clinically-possible/probable/definite IP (other criteria as for probands). Proband and 'spouse' lymphocyte subset data were contrasted with summary statistics (kit manufacturer's and published [34]) for haematologically-normal adult donors. The following were explored as determinants of FBC and subset profiles: serum haematinic and homocysteine concentrations (probands); serum autoantibody screen (probands); Helicobacter indices and results of hydrogen-breath-testing for SIBO (probands/spouses). In a subgroup, within-proband time-trends in cell counts (yearly sampling) were contrasted between the ureabreath-test (UBT) positive for Helicobacter infection and the UBT-negative, any impact of H. pylori eradication being noted. Any modifying effect of prevalent autoantibodies on the outcome of H. pylori eradication therapy was explored. This involved duplicate gait assessments on two baseline occasions and at ≥ 6-weekly intervals posttherapy, any background anti-parkinsonian medication being constant, and usage of levodopa being an exclusion.
In both phases, any anti-parkinsonian medication and laxative use were recorded. Protocols had been approved by local ethics committees, written consent obtained from participants.

Serum haematinics and homocysteine
Sera were stored at -20°C, and randomly allocated withinassay, with (except for Helicobacter serology, see below) between-assay stratification for subject-group, age and gender.

Serum immunoglobulin classes and autoantibodies
Immunoglobulin classes were measured by immunoturbidity (Cobas Mira Plus, Roche Diagnostics Corp., Indianapolis, USA). Inter-assay CV was 6.5 and 4.9% at 7.5 and 11.6 g/l IgG, respectively; 5.9 and 4.4% at 1.3 and 2.5 g/l IgA; and 4.5 and 3.4% at 0.7 and 1.1 g/l IgM. IgA subclasses were measured by radial immunodiffusion (Bind A Rid, Birmingham, UK): CV was 11% at 3360 mg/l IgA1; and 7.5% at 360 mg/l IgA2. Serum was screened for antinuclear, anti-smooth muscle, anti-mitochondrial, antigastric parietal cell and anti-liver/kidney microsomal antibodies. The additional autoantibodies listed, as well as the above, were sought before H. pylori eradication therapy and on average 1 1/2 years after, in a subgroup: antineutrophil cytoplasmic (ANCA), anti-thyroid peroxidase, anti-adrenal, anti-tissue transglutaminase, anti-intrinsic factor, anti-acetylcholine receptor, anti-neuronal nuclear and anti-purkinje cell. For each assay, samples were processed in a single batch, blind to sequence.

Helicobacter status
Helicobacter-status was defined by:-(i) [  Bland-Altman contrast of difference in and average of lym-phocyte counts (×10 9 /l) by two methods in 163 subjects confirmed in probands by endoscopic-biopsy for histopathology, culture and molecular-microbiology [23], eradication judged by UBT and, wherever possible, repeat biopsies.

Hydrogen-breath-test for small-intestinal-bacterialovergrowth
Hydrogen-breath-tests were carried out in UBT-negative probands and spouses, using a 25 g lactulose test-dose, following 24-hour deprivation of dairy products (and medicinal lactulose) and a breakfast of 250 ml black tea/ coffee or water. Hydrogen concentrations were measured (Micro Medical Ltd., Rochester, UK) pre-dose and at 15 minute-intervals for 4 hours after. A prolonged test, with non-absorbable substrate, was used because of potentially impaired gastric-empting and intestinal-transit [9,24]. Breath-hydrogen/time curves [36,37] were not bimodally distributed between-subject. Within-subject, two distinct peaks, early and 'colonic', were not present. Summary outcomes were examined: 2 hour value; maximum-value between 15 and 120 minutes; linear increase over 2 hours, when trend was to peak. Correlation coefficients were high for all contrasts, the maximum-value selected for analysis.

Statistical analysis
'Contemporaneous' reference range limits, constructed from a measurement made in 400 controls, would have a small standard deviation (approximately 0.09s, where s is standard deviation in the measurement), and be only marginally larger (0.1s) using 200 measurements [38].
In phase-2, controls below age 46 years had anomalous age-trends in FBC indices: these (19, presumably relatively-ill, individuals) were excluded. Since phase-2 controls were not subject to the rigorous inclusion/exclusion criteria of phase-1, their white cell counts were examined for evidence of distinct Gaussian distributions (e.g. a subgroup with a higher mean count, compatible with acute intercurrent infection). There was a tendency for two distributions to describe total white cell count better ( Figure  2), but no evidence that a mixture of distributions provided a better fit for lymphocytes or neutrophils. Comparisons with a 'conventional' reference range allowed robustness of any shift in distribution between subjectgroups to be tested. Wilcoxon signed-rank test was used to assess a shift from the reference median, chi-square goodness-of-fit to test whether the expected 5% of measurements fell outside the range.
Many chronic diseases are multi-step and multi-factorial: explanatory models were used to examine between-group and within-subject differences in laboratory measurements. Potential confounding or effect-modifying variables were assessed, examining for interactions prior to direct effects.
Where indicated, logarithmic transformations were employed, to ensure validity of assumptions of normality Distribution of total white cell count, and its decomposition into two Gaussian distributions Figure 2 Distribution of total white cell count, and its decomposition into two Gaussian distributions. Histogram (a) shows log e transformed raw data. Histograms (b) and (c) are generated from parameter estimates obtained by applying a Gaussian mixture model to log e (white cell count). They represent the 'best' mixture to replicate the overall distribution: i.e. two distributions tended to provided a better fit (likelihood ratio test, 3 degrees of freedom, χ 2 = 7.25, p = 0.06), (b) representing 80% of data, (c) a shift-to-the-right. There was no evidence that a mixture of Gaussian distributions provided a better fit to distribution of log e transformed lymphocyte (age-corrected) or neutrophil counts (χ 2 = 0.74 & 3.49, p = 0.9 & 0.3, respectively). of distribution and equality between groups of residual variance. The regression parameter for a log transformed outcome-variable was exponentiated, giving a relative (percentage) rather than absolute difference. That for a log transformed predictor was scaled by 1/log e 2, so that a unit change reflected a doubling. Table 2 shows that the major differences in FBC of IPprobands (P) and their spouses (Ps) from controls (C) were in total lymphocyte count. Mean count was lower by 23 In the 37.0% of probands never exposed to antiparkinsonian medication, mean lymphocyte count was 101.0 (95% CI: 89.5, 113.9)% of that in Ps, still significantly lower than in C. Exposure was associated with a 11.1 (0.1, 20.8)% lower count (p = 0.048), but inclusion of time-from-diagnosis (not important independently) reduced its significance to 0.1. Thus, medication status and time-from-diagnosis were intrinsically linked, possibly surrogate for a more pertinent measure of disease.

Full blood count
Neutrophil polymorphonuclear count tended to be higher in P (p = 0.1, gender adjusted) than in C. Haemoglobin concentration, red blood corpuscle count (RBC), packed cell volume (PCV), mean corpuscular volume (MCV), platelet count and mean platelet volume were not different in P or Ps from C. Mean corpuscular haemoglobin concentration (MCHC) was less in both than in C, by small but highly significant amounts (0.46 (0.27, 0.65); 0.33 (0.09, 0.58) g/dl, respectively, p < 0.001 & = 0.008, age and gender adjusted). Mean corpuscular haemoglobin (MCH) tended to be less in P (p = 0.06).

Lymphocyte subsets
Subset counts in 106 of P and 70 Ps, expressed as a percentage of subset sum, are compared with the kit manufacturer's reference mean and 95% confidence limits in Table 3 and Table 4. Figure 4 summarises direction of effects. In P, CD4+ and CD8+ distributions were platykurtic, greater proportions than expected falling both above and below reference ranges. In Ps, there was a significant rightward shift in mean CD4+, and a significant leftward shift in CD8+ with respect to lower reference limit. A sig-nificant leftward shift in mean CD19+ was seen in both P and Ps. That to-the-right in mean CD16+56+ characterised P alone. Subset shifts were not explained by anti-parkinsonian medication.
Regarding within-couple concordance (70 pairs), CD3+, CD4+, CD8+ and CD19+ counts were lower in probands, by 14.2 (2.0, 24.9), 18.2 (5.1, 29.5), 17.1 (2.1, 29.8) and Contrast of lymphocyte counts (×10 9 /l) in probands with idiopathic parkinsonism and their spouses with an age-specific 95% reference range based on routine requests Figure 3 Contrast of lymphocyte counts (×10 9 /l) in probands with idiopathic parkinsonism and their spouses with an age-specific 95% reference range based on routine requests. Counts are corrected as if all subjects were male, using the relationship to gender in controls, there being no evidence of an age.gender interaction.  Table 4) of 56-86%, in their spouses with range but not mean.  Figure 5 illustrates that the long thin duodenal enterocyte mitochondria found in IP are not necessarily associated with abnormal lymphocyte mitochondrial morphology.

Serum haematinic and homocysteine concentrations
Mean nutrient intakes were estimated from 3-day food diaries in 23 of the phase-2 probands: they complied with UK National Reference Values.
In all 126 probands (Table 2), serum homocysteine was above the desired-maximum of <16 μmol/l in 43.2 (binomial exact 95% CI: 34.1, 52.7)%. Figure 6 shows that the expected proportion had a serum B12 below reference range (<180 ng/l), but 16 Six percent of probands were both newly diagnosed and untreated: their median homocysteine concentration, 13.4 μmol/l, was below the desired maximum. Receiving anti-parkinsonian medication other than levodopa Shifts in percentage distribution of lymphocyte subsets in probands (P), with idiopathic parkinsonism, and their spouses (Ps) compared with reference range (a) mean and (b) its lower and upper reference limits Figure 4 Shifts in percentage distribution of lymphocyte subsets in probands (P), with idiopathic parkinsonism, and their spouses (Ps) compared with reference range (a) mean and (b) its lower and upper reference limits. In P (black bar) and Ps (grey bar), significant differences are denoted by black star and grey star, no subject outside reference limit by black square and grey square. In (b), more than the 2.5% expected had CD4+ (in P), CD8+ (P & Ps) and CD19+ (P) below lower limit (exact binomial test: p ≤ 0.01 in each case), and CD4+ (P & Ps), CD8+ (P) and CD16+56+ (P) above upper limit (p ≤ 0.02).
Serum ferritin was below the reference range (20-300 μg/ l males; 20-200 μg/l post-menopausal females) in 6.8 Neither homocysteine nor these haematinics contributed to explaining total lymphocyte count in IP.

Prevalence of autoantibodies
Anti-nuclear antibody (ANA) was present in 25.3% of the 126 phase-2 probands, anti-intrinsic factor in 15.1%, and anti-gastric parietal cell in 7.6%. Gastric autoantibodies were not associated with a lower serum B12. Anti-smooth Lymphocyte and duodenal enterocyte ultrastructure in idiopathic parkinsonism (IP) Figure 5 Lymphocyte and duodenal enterocyte ultrastructure in idiopathic parkinsonism (IP). Electron micrographs shows mitochondria in a representative blood lymphocyte from (a) a man with clinically-definite lP and (b) an age-matched healthy man, and in a representative duodenal enterocyte from (c) this IP-proband (panel to right shows higher magnification) and (d) his spouse. The proband's normal lymphocyte mitochondria are in marked contrast to his and his spouse's long thin duodenal mitochondria. The proband's IP had been diagnosed 7 years previously. He was receiving anti-parkinsonian medication. His spouse had probable-IP on screening. Both had bloating and cyclical diarrhoea going back 10 years. Typically, in the proband, 4 days of unformed stool alternated with constipation in a four-week cycle. In the spouse, explosive watery diarrhoea followed abdominal cramps, over 2 days in 2 week cycles, with normal bowel habit in between. Both had a positive hydrogen-breath-test (criterion: two consecutive values [37] >cut-point of meter manufacturer), and were negative for Helicobacter by UBT, serology, and culture/molecular microbiology on gastric biopsy. http://www.gutpathogens.com/content/1/1/20 muscle antibody was present in 2.6%, anti-mitochondrial and anti-liver/kidney microsomal negative throughout. In the more comprehensive screen in 21 probands, ANCA was present in 3, but none had antibodies against mye-loperoxidase or proteinase-3 targets. Total lymphocyte count was not associated with autoantibody-status. Table 5 shows that, in phase-1, serum immunoglobulin concentrations in the 120 IP-probands and 205 contemporaneous controls were similar. Figure 7 shows that presence, in controls, of hand-bradykinesia (16%), or postural abnormality (9%), was very strongly associated with a higher IgG and IgA. Stridelength was shorter by 59.3 (95% CI: 1.9, 116.8) mm if IgG concentration doubled (p = 0.04), but gait was not associated with IgA. Resting rigidity in arms was rare (1%), but rigidity evoked by movement of the contralateral limb, the activation phenomenon, common (70%) and associated with IgA (Figure 7 footnote). Resting tremor was also rare (1.5%), postural tremor common (48%) but not associated with immunoglobulin classes. Of these features, only hand-bradykinesia was associated with IgM, by contrast a strong negative relationship. All associations with IgA were attributable to IgA1.

Serum immunoglobulin class concentrations
Immunoglobulin concentrations, in probands, were unrelated to the presence/absence of a cardinal feature or stride-length. Concentrations were not associated with Relationship of serum homocysteine to B12 concentration in idiopathic parkinsonism Figure 6 Relationship of serum homocysteine to B12 concentration in idiopathic parkinsonism. Two probands with exceptionally high homocysteine (110 and 135 μmol/l) are excluded: the first had a low B12 but normal folate, the second (with frank H. pylori infection and an empyema) a low folate with a normal B12. Neither had received levodopa.

Helicobacter-status of spouses and probands
Crude prevalence rate estimates for 'Parkinson's disease' in 34 European studies range widely, from 65.6 to 12,500 per 100,000 [39]. The estimate lay within the binomial exact 95% CI for the spouse cohort (9,195 (4,045, 17,317) per 100,000) in only one study. All 8 spouses with clinically-definite IP had 'frank' UBT-positive Helicobacter infection, only 8% of the other spouses did (P < 0.0001). Table 6 shows that frequency of positive Helicobacter-status, by breath-test or serology, tended to be less in the spouse cohort than in presenting cases.
In the earlier IP-cohort, prevalence of seropositivity was not significantly different from that in the socially-similar controls ( Table 6). The odds of a proband's sample being immunoblot-positive and ELISA-negative, compared with vice versa, became 69.1(95% CI: 4.9, 978.3) times greater in phase-2 than, 10 year earlier, in phase-1 (p = 0.02). In the later IP-cohort, ELISA-positivity was shown to repre-sented frank infection, in that its prevalence concurred with that of UBT-positivity.

Helicobacter and blood profile Cellular profile
In the later IP-cohort and their spouses (Table 6), UBTpositivity was not associated with total lymphocyte count (nor was ELISA-positivity). However, the CD8+ subset count was higher with frank infection by 27.8 (6.1, 53.9)%, (p = 0.01, age adjusted: no interaction between presence/absence clinically-definite IP and Helicobacterstatus, nor any direct disease-status effect). Immunoblotpositivity was much more frequent and associated with a higher total lymphocyte count (by 11 Serial lymphocyte counts were obtained in 49 probands (203 observations, median 4 (interquartile range 3, 5), over 2.6 (2.1, 3.5) years), counts were stable in the 21 without frank infection. However, following biopsyproven H. pylori eradication in 28, lymphocyte count did tend to increase (3.6 (95% CI: 0.0, 7.8)% per year, p = 0.08), there being no prior time-trend.

Autoantibody-status
Neither UBT-nor immunoblot-status influenced the frequency of ANA, the most common autoantibody found in clinically-definite IP. in contrast (p = 0.03) to improvement in the ANA-negative (77 (-1, 156) mm/year). Anti-nuclear antibody appeared to mark a poor prognosis, but effects of ANAstatus and success/failure of eradication on stride-length were intrinsically linked. A lower CD8+ or double-positive CD4+CD8+ count was also associated with deterioration in stride-length (p = 0.02, 0.03 respectively, age/ gender adjusted as appropriate), and tended to be with eradication failure (p = 0.06, 0.04). Indeed, a lower CD8+ count appeared linked with ANA-positivity (p = 0.07). Neither CD16+56+ count nor serum homocysteine were prognostic indicators in this context.

Immunoglobulin class concentrations
Serum IgM concentration was lower with ELISA-positivity in phase-1 controls (Table 5, footnote), but not in clinically-definite IP (p = 0.02, for subject group.Helicobacterstatus interaction). This interaction also obtained when Helicobacter-status was defined by the immunoblot, in particular by the presence/absence of a single (47 kDa) band (p = 0.008). Notably, the same band explained the immunoblot association with IgM in controls (p = 0.001).
There was no evidence that other infections, consequent on functional impairment, were responsible: IgM was not higher the greater the Hoehn and Yahr Staging of IP [40].
No effect of subject group on other immunoglobulin classes was found, relative to Helicobacter serology or directly.
In controls, Helicobacter serology explained neither the relationship of features of clinically-possible/probable IP to immunoglobulin concentration (Figure 7) nor their presence directly.

Small-intestinal bacterial-overgrowth and blood profile
Maximum breath-hydrogen between 15 and 120 minutes (median 41 (interquartile range 17-73) ppm) was not influenced by age, gender or subject group (40 probands, 15 spouses without clinically-definite IP, in phase-2). The meter manufacturer's diagnostic cut-point (20 ppm increment) was exceeded at least once within the 2 hours in 65% (irrespective of subject-group), by two consecutive readings [37] in 60%.
Although the maximum was not associated with total lymphocyte count, it was with the CD4+ subset count (estimated increase 9.2 (95% CI: 1. Probands' folate, B12, homocysteine and autoantibodystatus were unrelated to maximum breath-hydrogen. Probands' and spouses' CD4+/CD8+ ratios were not explained by breath-hydrogen (nor UBT-status or immunoblot-status).
Helicobacter infection appeared to keep SIBO at bay. Breath-hydrogen was higher (Spearman rank correlation, p = 0.003) in those of the currently UBT-negative probands and spouses who had been UBT-positive on presentation. It tended to be higher (p = 0.06) where the immunoblot score had been positive/equivocal. Hydrogen-breath-tests were performed, in two subsequent probands, before and serially after biopsy-proven eradication of Helicobacter (initially detected by molecular-microbiology on two culture-negative biopsies). One had become positive (two consecutive values criterion) at 8 months, the other converted between 12 and 18 months: even low-density infection appeared protective. Table 7 summarises individual clues yielded from the blood profiling with reference to a 'premonitory' stage, and prognostic indicators within established IP. Corresponding roles for pathogens in the evolution are postulated on the basis of the background, results and discussion below.

Discussion
Reduced total lymphocyte count in clinically-definite IPprobands (even when never exposed to anti-parkinsonian medication) and their spouses is a robust finding. Suppression of T-cell proliferation by Helicobacter [27,28] does not appear responsible. There was no cross-sectional association between lymphocyte counts and Helicobacterstatus, although probands' total count tended to increase following eradication. No relationship was found between total count and hydrogen-breath-test result, but the impact of SIBO eradication/reacquisition was not investigated. Generalised autoantibody seropositivity would have supported an autoimmune pathogenesis for IP: its absence cannot exclude. Whilst dysfunction of lymphocyte mitochondria is reported in IP [31], they do not necessarily exhibit the abnormal morphology seen in enterocytes. Haematinics, or immuno-inflammatory activation as flagged by homocysteine [41], did not account for the reduced total count.
Unexplained reduction in lymphocyte count and prodromal enteric-nervous-system damage [19,20] might be viral in origin. Whereas ascent via gastrointestinal neural pathways [8] would fit with 'cold neurodegeneration', dissemination in blood could explain constitutional symptoms and widespread pathology. B-cells could be a target (as with Epstein-Barr virus): their proportion was reduced in spouses, more so in probands. Natural-killer cells provide first-line viral defence [42]. Probands had an increased proportion, whereas spouses had an increase in CD4+. Given the same insult, this suggests a difference in immune competence/regulation. Cytotoxic T-cells have an important role in eliminating virus-infected cells. Their platykurtic proportional distribution in probands may encompass an increase in earlier stages (as in asymptomatic human immunodeficiency virus, HIV, infection) and suppression in later stages (as in acquired immunodeficiency syndrome, AIDS). A low CD4+/CD8+ ratio can flag viral infection, classically HIV [34], and has been reported in IP [4,5]. Here, addressing age/gender covariates, it was low only in younger male probands and spouses.
We have classified intracellular microbial targets in IP for current diagnostic assays [18]. These include enteroviruses, which infect via the gastrointestinal tract and can have neurological consequences. In post-poliomyelitis syndrome, there is systemic illness with raised serum inflammatory markers, and metabolic and skin involvement [43][44][45]. Parkinsonism is reported [46], lymphopenia not. The century-old proposal linking poliomyelitis with motor-neurone disease has been updated to encompass subclinical infection [47]. Increasing mortality from motor-neurone disease, where vaccination has triumphed over clinical poliomyelitis [48], does not preclude this. Searching for undiscovered viruses requires leads. The epi- demiology of IP and HIV are distinct, but parkinsonism is seen in uncomplicated HIV-infection (not just with opportunistic infections in AIDS), and jejunal autonomic denervation described [18]. Thus a relatively benign retrovirus might explain the lymphocyte profile and predisposition to SIBO in IP-probands and spouses.
The relative lymphopenia in spouses complements our finding of marked differences (physiological/psychomotor/dermatological) relevant to parkinsonism between spouses, cohabiting with probands for half-a-century, and control couples [12,33,49,50]. Those spouses were, on average, a short, albeit highly significant, 'distance-downthe-pathway'. Their multifarious manifestations are difficult to dismiss as selective mating or learned/reactive behaviour. Here, both probands and spouses had a high frequency of hydrogen-breath-test-positivity. Indeed, we have estimated that 53% of probands and 36% of spouses have chronic bowel dysfunction by stringent definition [51]. Seven percent of probands, 14% of spouses have diarrhoea (unformed stool during ≥3/4 past year, plus ≥3 bowel movements/day for 1/2). Greater enteric (± vagal nucleus) neuronal damage in probands may stave-off a diarrhoeal response to SIBO. The implication is of a 'premonitory' stage in spouses, alone lacking impetus to reach the diagnostic threshold. Lack of within-couple concordance in Helicobacter-status between probands and spouses [23] suggests that any 'conjugally-transmitted primer' is not Helicobacter. However, transmission of a 'primer' to a Helicobacter-colonised recipient may result in progression to the diagnostic threshold. Both might be necessary, neither sufficient. Indeed, here, spouses had a relatively high prevalence of clinically-definite IP, and all so affected had frank Helicobacter infection. Persistence and burden of Helicobacter in established IP are discussed below.
Strong associations of sporadic cardinal features of parkinsonism with changes in circulating immunoglobulins also indicate a premonitory active process. That IgA associations were accounted for by IgA1 may indicate a bone marrow response, but does not exclude a mucosal [52]. The apparent link of constipation with IgA1 in probands suggests the latter. Frequency of defaecation begins to deviate from that of controls three decades before the median age of neurological diagnosis [20]. Moreover, a higher IgA1 was associated with activation of rigidity, a common sign in our controls, which enables "one to detect Parkinson's disease in its earliest phase" [53]. Helicobacter serology did not explain immunoglobulin associations, a virus and/or SIBO might. We describe a differential IgM response to Helicobacter between controls and probands. In IP, sequestration to the site of inflammation may no longer obtain, or there be increased production of poly-specific IgM by naïve-B-or B1-cells, despite the lower overall proportion of B-cells.
Hyperhomocysteinemia is associated with cardiovascular risk and conditions which overlap with parkinsonism: dementing illnesses [54] and depression [55]. Moderate elevation of homocysteine in nearly half of the IPprobands was explained only in small part by serum B12, with no complementary effect of folate. Hyperhomocysteinemia was unrelated to Helicobacter-status: absence of even moderate atrophy characterizes Helicobacter-associated gastritis in IP. [23,24]. The underlying cause is likely to be SIBO, in which there is increased bacterial utilisation of B12. We propose that consequent immunoinflammatory activation drives homocysteine production [41]. Activation may increase demand for B12, such that a concentration in the 'equivocal' range should be regarded as pathological. An inflammatory response to SIBO would fit with the inverse association between breath-hydrogen and indicators of iron-absorption. Interleukin-1β, for example, acts centrally, as well as on gastric receptors, to inhibit acid secretion and hence iron-absorption [56]. Indeed, exploratory studies suggest a greater prevalence in IP of cytokine-gene polymorphisms predisposing to an intense innate inflammatory response [57]. Suppression of inflammation can reduce homocysteine effectively in rheumatoid arthritis [41], but this approach is precluded where the source is infection. In IP, eradication of SIBO for several years is feasible, where adequate gastrointestinal-transit can be maintained by sufficient fluid-intake, a high-fibre diet and bulk/osmotic laxatives [18].
There is no evidence that hyperhomocysteinemia in IP is genetically determined [58]. Cognitive impairment, clinical depression, other physical incapacity and cardiovascular symptoms were exclusions [54,55]. Hyperhomocysteinemia has been attributed to levodopa: its O-methylation by catechol-O-methyltransferase (COMT) can provide a substrate for homocysteine synthesis [59]. We clearly demonstrate that the only relevance of levodopa (in modest doses) is its surrogacy for time-fromdiagnosis. Moreover, neither reducing/stopping levodopa, nor inhibition of its peripheral wastage by co-prescription of a COMT-inhibitor, reduces homocysteine [59].
Anti-nuclear antibody, present in a quarter of probands, was an indicator of the outcome of anti-Helicobacter therapy. Outcome is uncertain in IP even when anti-microbial sensitivities are known/compliance is monitored [23,24]. Gait improves in IP following 'biopsy-proven' H. pylori eradication [18,23,24]. Here, following anti-Helicobacter therapy, gait deteriorated in the ANA-positive in stark contrast to performance in the ANA-negative. ANA appeared to alert to continuing infection, and might be more sensitive to its detection than examining two biopsies. Bolus antigen release may accentuate deterioration where anti-Helicobacter therapy fails. ANA-positivity and a lower CD8+ count were intrinsically linked. Reduced cytoxicity may have impaired clearance of residual organisms. Compensation by expression of CD8 on mature CD4+ cells [60] may have failed, even though an increased proportion of such cells (CD4bright+CD8dull+) has been reported in IP as a whole [5,6]. Although NK-activity is stimulated by H. pylori in vitro [61], the NK-count was not a prognostic indicator.
The increase, over a decade, in immunoblot-positivity over ELISA-positivity may signify more low-density and occult (gastric lymph node [62]) infection. It may or may not be IP-specific. Association of a higher lymphocyte count with immunoblot-positivity suggests that it represents infection rather than memory. More low-density infection would fit with greater incidental antimicrobial use in a context of impaired bacterial clearance. Propensity to hidden infection/resting (coccoid) forms might be greater where the 'protective' urease continues to produce ammonia in the face of cytokine-driven inhibition of gastric acid [56]. Such progressive hypochlorhydria could explain peptic ulcer being prodromal by decades in IP [26], and the increased likelihood of seropositivity in younger probands [63]. That Helicobacter detected only by molecular-microbiology on gastric biopsies appears to offer the same therapeutic opportunity as does frank infection [18], underlines its relevance to IP. Moreover, persistence at this level following treatment of frank infection appears detrimental [23]. These findings may predicate on an autoimmune response to Helicobacter.
In conclusion, the explanation of the different manifestations of IP between-probands, and within-proband over time, may lay in the interaction between more than one pathogen and determinants, genetic and environmental, of inflammatory response. Helicobacter may be necessary though not sufficient, SIBO a frequent, relatively non-specific and dose-related, player. A viral premonitory phase may prove the common denominator for IP, other 'neurodegenerative' diseases and neuro/psychiatric syndromes and 'functional' gastrointestinal disorders.