Additional file 4: Table S2. of Multifocal gastric adenocarcinoma in a patient with LRBA deficiency

Selected immune system determinants longitudinally evaluated in the patient. (DOCX 15 kb

Additional file 5: Figure S3. of Multifocal gastric adenocarcinoma in a patient with LRBA deficiency

Ultrasound and histopathologic images of the gastric cancer. Description: (a.) Ultrasound image of a 49 × 44 mm large tumor formation in the stomach; (b., c.) histopathologic images of bioptic material from the gastric tumor demonstrating intestinal type gastric carcinoma with lamina propria invasion (Kreyberg trichrom stain, 20X magnification (b.) 100X magnification (c.)). The square in part b. indicates the area of magnification in part c. of the figure. (PDF 223 kb

Additional file 2: Figure S2. of Multifocal gastric adenocarcinoma in a patient with LRBA deficiency

The identified regions of homozygous stretches in chromosome 2, 5 and 6. Description: Blue regions indicate homozygous variants and yellow regions indicate heterozygous variants. Orange regions in parental chromosomes (F, M) indicate heterozygous variants corresponding with homozygous variants of sibling’s genotype (P). (TIFF 898 kb

Additional file 3: Table S1. of Multifocal gastric adenocarcinoma in a patient with LRBA deficiency

Autoantibodies evaluated in the patient. (DOCX 12 kb

Additional file 1: Figure S1. of Multifocal gastric adenocarcinoma in a patient with LRBA deficiency

Workflow of WES filtering process with multiple filtering steps including a phenotype driven filtering. (PDF 308 kb

Morphological and flow cytometry profile results of prolonged limbal explant cultures cultivated without amniotic membrane.

<p>Limbal explant outgrowth of cells cultured on plastic culture plates in the absence of feeder layer cells and in medium supplemented with only human serum showed after 3 weeks of culture two morphologically distinct cell population types, a predominantly epithelial-like (black arrow) and a more fibroblast-like cell population (white arrow) (A). A sphere-shaped formation with spindle shaped cells around it was observed (B). (A,B: Magnification:4x). A limbal explant culture after 2 months of culturing without any passaging showing a translucent corneal stromal-like 3D tissue (C) (the total time of observation in culture was 6 months). A plot representing the mean percentages ± SEM (n = 7, paired two-sample t-test) of flow cytometry profiles, red bars representing the primary limbal cultures and white bars the transfered secondary limbal cultures, the differences not being statistically significant (p > 0.05) (D). (*) means limbal explant; (**) means translucent tissue. Bar, 100 μm.</p

Limbal explant outgrowth of cells cultured on amniotic membrane after 3 days of culture and after 3 weeks of culture.

<p>The cells adjacent to the limbal explants were more uniform, smaller and had larger nuclei (A). In the first week of culture an epithelial line (B) was observed on AM (**), which after 3 weeks of culture reached the AM border (C) (◆) (A,B: Magnification:10x; C: Magnification:4x). Light microscopy showed limbal epithelial cells cultivated on AM (stromal side) produced a well-stratified cell layer (D); (HE: 10x). (*; explant; AM: amniotic membrane; HE: hematoxylin and eosin). Bars, 100 μm.</p

Immunohistochemical comparison of primary and secondary limbal explant cultures cultivated on the epithelial or stromal side of the AM in a human serum supplemented medium.

<p>In primary limbal explant cultures cultivated for 14 days, limbal epithelial growth was observed on both sides of the AM on HE sections (A), similarmorphologywas observed in secondary limbal explant cultures on both sides of the AM after the limbal explants were cultured for 40 days on plastic culture plates and stained positive for pan-cytokeratinmarker after 21 days of culture on AM (B). Cells from primary and secondary cultures on both sides of the AM (explants transferred after 14 days, from the same donor) expressed p63 and Ki67 marker, however in the secondary cultures a slight decrease was observed for Ki67 marker expression (C). (Magnification A:20x, B:10x; C:20x). (AM: amnioticmembrane; epithelial side, + stromal side; HE: hematoxylin and eosin). Bars, 50 μm.</p

Results of flow cytometry profiles of confluent primary limbal explant cultures cultivated on plastic culture plates in only 10% or 20% human serum supplemented medium.

<p>A plot of the mean percentages of positive cells ± SEM for the tested markers in both tested HS concentration groups is shown (Student two-tailed t-test; * p < 0.05).</p

DataSheet_1_Alterations in immunophenotype and metabolic profile of mononuclear cells during follow up in children with multisystem inflammatory syndrome (MIS-C).pdf

IntroductionAlthough children seem to be less susceptible to COVID-19, some of them develop a rare but serious hyperinflammatory condition called multisystem inflammatory syndrome in children (MIS-C). While several studies describe the clinical conditions of acute MIS-C, the status of convalescent patients in the months after acute MIS-C is still unclear, especially the question of persistence of changes in the specific subpopulations of immune cells in the convalescent phase of the disease.MethodsWe therefore analyzed peripheral blood of 14 children with MIS-C at the onset of the disease (acute phase) and 2 to 6 months after disease onset (post-acute convalescent phase) for lymphocyte subsets and antigen-presenting cell (APC) phenotype. The results were compared with six healthy age-matched controls.ResultsAll major lymphocyte populations (B cells, CD4 + and CD8+ T cells, and NK cells) were decreased in the acute phase and normalized in the convalescent phase. T cell activation was increased in the acute phase, followed by an increased proportion of γ/δ-double-negative T cells (γ/δ DN Ts) in the convalescent phase. B cell differentiation was impaired in the acute phase with a decreased proportion of CD21 expressing, activated/memory, and class-switched memory B cells, which normalized in the convalescent phase. The proportion of plasmacytoid dendritic cells, conventional type 2 dendritic cells, and classical monocytes were decreased, while the proportion of conventional type 1 dendritic cells was increased in the acute phase. Importantly the population of plasmacytoid dendritic cells remained decreased in the convalescent phase, while other APC populations normalized. Immunometabolic analysis of peripheral blood mononuclear cells (PBMCs) in the convalescent MIS-C showed comparable mitochondrial respiration and glycolysis rates to healthy controls.ConclusionsWhile both immunophenotyping and immunometabolic analyzes showed that immune cells in the convalescent MIS-C phase normalized in many parameters, we found lower percentage of plasmablasts, lower expression of T cell co-receptors (CD3, CD4, and CD8), an increased percentage of γ/δ DN Ts and increased metabolic activity of CD3/CD28-stimulated T cells. Overall, the results suggest that inflammation persists for months after the onset of MIS-C, with significant alterations in some immune system parameters, which may also impair immune defense against viral infections.</p