None of the individuals enrolled in the study had suffered from HSV-1 encephalitis in the past; no evidence of a particularly severe herpes labialis was recorded in the anamnesis. inefficient IgG3 humoral immune response, failing to block viral replication, contributes to progressive neurodegeneration. gene [20]. 2.4. Morphometrical AnalysesMRI To investigate gray matter morphometry, in close temporal proximity with blood sampling, a randomly-selected subgroup of AD (= 40) and MCI (= 35) individuals underwent a high-resolution 3D-T1 image acquired on a 1.5 Tesla scanner (Siemens Magnetom Avanto, Erlangen, Germany). Parameters of the high-resolution 3D-T1 (MPRAGE) image were TR/TE = 1900/3.37 ms, FoV = 192 mm 256 mm, in-plane resolution 1 mm 1 mm, slice thickness = 1 mm, and number of contiguous axial slices = 176. 3D-T1 images were analyzed using Freesurfers recon-all pipeline (v 5.3, https://surfer.nmr.mgh.harvard.edu/ [21]. Quality checks were performed according to the manual quality control procedure described in [22] and corrections were manually performed to improve automatic segmentation. In order to obtain total hippocampal volumes, the hippocampal subfield segmentation tool of Freesurfer (v.6.0) [23] was used and estimated total intracranial volume (eTIV) was also computed using Freesurfer automatic subcortical segmentation [24]. To obtain gray matter atrophy measures, cortical parcellations were obtained for each subject according to Desikan atlas [25] and were used to obtain three ad hoc brain masks, created according to literature (see Figure 1). These masks were: (1) brain mask of AD target areas in the mild stages of the disease (AD-mask), including bilaterally-posterior cingulate cortex and temporal lobe areas (superior, middle, and inferior gyrus, medial temporal lobe areas, and total hippocampal and amygdala volumes) [26,27]; (2) brain mask of HSV-1 target areas (HSV-mask), including bilaterally-anterior cingulate and orbitofrontal cortices, medial temporal lobe areas, insula and total hippocampal, and thalamus and amygdala volumes according to [28,29,30,31]; and (3) a mask including the brain regions overlapping between AD-mask and HSV-mask brain masks (ADHSV-1-mask), including bilaterally-medial temporal lobe cortices and total hippocampal and amygdala volumes. Open in a separate window Figure 1 Ad-hoc masks of brain areas considered in the statistical analyses. List of all the brain areas included in each mask: in red color, brain regions that are target areas in the mild stages of AD pathology (AD mask); in yellow color, cortical areas that are the target for HSV-1 encephalitis (HSV-mask); and in orange color, the areas commonly shared by AD mask and HSV-mask (ADHSV-1-mask). AD: Alzheimer disease; C: cingulum; F: frontal; TL: temporal lobe, lateral aspect; TM: temporal lobe, medial aspect; I: insula; P: parietal; O: occipital; SUB: subcortical volumes. Morphometrical data (volumes/thickness) were extracted for each subject and included in subsequent statistical analyses: (a) to test downstream neuronal degeneration in our sample and (b) to test the relationship between morphometrical features and IgG3 Ab titers (see statistical analysis section). 2.5. Statistical Analysis 2.5.1. Demographical and HSV-1-IgG Analyses The parametric data are expressed as mean standard deviation, whereas the non-parametric data are expressed as median and interquartile range (IQR). Differences in experimental data among groups were tested using the KruskalCWallis TSHR test and, when appropriate, the MannCWhitney U test, and the correlations, using Spearmans correlation coefficient. 0.05 statistical threshold. (b) To test the presence of relationship between Clinofibrate IgG3 titers and morphometrical indices, statistical analyses were performed separately for AD and MCI subsamples considering hippocampal volumes and cortical thickness in the selected areas (AD mask, HSV-mask, ADHSV-1-mask) and including only those subjects with serum Clinofibrate presence of IgG3 = 30 MCI and = 27 AD). Partial correlations between IgG3 Ab titers and brain thicknesses were then computed Clinofibrate using MedCalc software (v. 19.1). Age, gender, education, and mini mental state evaluation (MMSE) raw score were included as covariates of no interest. Partial correlations were also computed between IgG3 Ab titers and subcortical volumes (hippocampus, thalamus, and amygdala) also inserting eTIV as an additional covariate of no interest. Statistical analyses were considered as statistically significant when surviving 0.05 statistical threshold. 3. Results 3.1. Demographical and Clinical Characteristics of the Subjects Gender, educational level, and age were comparable in the three groups examined; global cognitive levels (MMSE) were, as per definition, significantly reduced in AD and MCI subjects compared to HC.
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