Supplementary Material for: Carotid Arterial Hemodynamic in Ischemic Levkoaraiosis Suggests Hypoperfusion Mechanism

<b><i>Background:</i></b> Leukoaraiosis (ILA) is believed to be ischaemic in origin due to its similar location as that of lacunar infarctions and its association with cerebrovascular risk factors. However, its pathophysiology is not well understood. The ischaemic injuries may be a result of increased pulsatility or cerebral hypo-perfusion. We used carotid duplex ultrasound to prove that the underlying mechanism is hypo-perfusion. <b><i>Methods:</i></b> We compared 55 ILA patients to 44 risk factor-matched controls with normal magnetic resonance imaging (MRI) of the head. ILA diagnosis was based on MRI and was further categorised according to the Fazekas scale. We measured carotid artery blood flow velocity and diameter and calculated carotid blood flow and resistance indexes. <b><i>Results:</i></b> Blood flow velocities and blood flows were significantly lower in the ILA group, including diastolic, systolic and mean pressures (p ≤ 0.05). The resistance indices were higher in the ILA group, but the differences were not statistically significant. All the velocities and blood flows showed a decreasing trend with higher Fazekas score, whereas resistance indexes showed an increasing trend. <b><i>Conclusions:</i></b> Lower blood flow and higher resistance of carotid arteries are consistent with the hypo-perfusion theory of ILA. Carotid ultrasound could have a diagnostic and prognostic role in ILA patients

Interfering substances evaluation with TK 210 ELISA.

(PDF)</p

Distribution of TK1 protein and TK1 activity values in sera from patients with breast cancer.

(A) TK1 protein values in sera from blood donors (●) and patients with breast cancer (■). (B) TK1 activity levels in sera from blood donors (●) and patients with breast cancer (■). (C) Comparison of TK1 protein levels in sera from blood donors (●), T1 (■), T2 (▲) and T3 (▼) breast cancer patients. (D) Comparison of TK1 protein levels in sera from blood donors (●), T1 (■), T2 (▲) and T3 (▼) breast cancer patients. (E) Comparison of TK1 protein levels in patients without metastasis (M0) (●) and patients with metastasis (M1+M2) (■). (F) Comparison of TK1 activity values in patients without metastasis (M0) (●) and patients with metastasis (M1+M2) (■). The error bars denote median.</p

TK 210 ELISA standard curve and assay linearity.

(A) A standard curve for TK 210 ELISA with calibrators (0–18 ng/mL) using 4-PL (non-linear regression program) in Graph pad prism. The limit of detection was 0.12 ng/mL. The error bars indicate the mean and standard deviation (SD) of 10 different experiments performed in duplicate. (B) The percentage change in calibrators from the precision assays. The error bars indicate the mean and standard deviation (SD) value of the calibrator. (C) The OD values of serially diluted serum samples measured by AroCell TK 210 ELISA. (D) The correlation between expected and observed TK1 protein levels in serially diluted samples.</p

TK1 protein and TK1 activity values in sera from patients with prostate cancer.

(A) TK1 protein values in sera from blood donors (●) and patients with prostate cancer (■). (B) TK1 activity levels in sera from blood donors (●) and patients with prostate cancer (■). (C) Comparison of TK1 protein levels in sera from blood donors (●), GS below 7 (■), GS 7(▲) and GS 8+9 (▼) prostate cancer patients. (D) Comparison of TK1 activity levels in sera from blood donors (●), GS below 7 (■), GS 7(▲) and GS 8+9 (▼) prostate cancer patients. The error bars denote median.</p

Dilution linearity of AroCell TK 210 ELISA.

Dilution linearity of AroCell TK 210 ELISA.</p

Age based comparison of TK1 activity, TK1 protein in breast cancer patients and prostate cancer patients.

(TIF)</p

The distribution of serum TK1 protein and TK1 activity in blood donor samples (n = 159).

(A) Serum TK1 protein distribution in blood donor sera (n = 159) (B) A comparison of TK1 concentrations in sera from blood donor men below 60 years (●) and men above 60 years (■). The error bars indicate the median (red). (C) Serum TK1 activity levels in blood donor samples (n = 159). (D) Comparison of TK1 activity in sera from blood donor men below 60 years (●) and men above 60 years (■). The error bars indicate the median (red).</p

TK1 protein and TK1 activity levels in sera from patients with hematological malignancies.

(A) TK1 protein distributions in sera from blood donors (●) and patients with hematological malignancies (■). (B) TK1 activity levels in sera from blood donors (●) and patients with hematological malignancies (■). (C) Comparison of TK1 protein levels in sera from blood donors (●), MM (■), CLL (▲), MDS (▼). and AML (▪). (D) Comparison of TK1 activities in sera from blood donors (●) MM (■), CLL (▲), MDS (▼). and AML (▪). The error bars denote median. (E) Correlation between TK 210 ELISA (ng/mL) and age (years) in HM patients. (F) Correlation between TK1 activity and age (years) in HM patients.</p

Characterization of TK1 antibodies.

(A) An SDS-PAGE was performed with a 4% - 20% gradient gel for analysis of purified recombinant TK1, followed by Coomassie Blue staining, Lane 1: BSA (2.00 μg), Lane 2: Thymidine Kinase 1 (2.00 μg). (B) Western blot analysis of His-tag purified recombinant TK1 using Anti-His antibody (0.2 μg/mL), Lane 3: Thymidine Kinase 1 (C). The amino acid sequence of the human TK1 cDNA. The red color indicates the peptide sequences used for TK1 antibody production. (D) A model of the full-length human TK1 tetramer (produced by Martin Welin et al ref 14). The exposed KEN sequence and the C-terminal are shown in red and purple, and the lasso loop/active site is shown in light blue. (E) Pepscan analysis of the binding site of the anti-TK1 monoclonal antibodies with peptides from the 193–226 regions of TK1.</p