PMC7726418_01
Case report timeline and assessment of the tolerability of systemically administered ADMSC (A) Temporal diagram representing major events, interventions and outcomes. Day 0 (first infusion): October 5, 2017, Day 21 (second infusion): October 26, 2017, Day 42 (third infusion): November 16, 2017, 3 months: November 30, 2017, 4 months: January 11, 2018, 6 months: April 9, 2018, 9 months: July 19, 2018; 1 year: October 4, 2018; 2 years: November 11, 2019. (B) Adverse reactions registered during the 12 h following all three infusions. (C) Hemostatic parameters measured just 2 h before (2hb) and 4 h after (4ha) the first infusion and 2 h before third infusion (2hb). Rotational thromboelastometry (ROTEM) parameters determined for EXTEM, INTEM, and FIBTEM tests are shown. EXTEM test, extrinsic pathway thromboelastometry; INTEM test, intrinsic pathway thromboelastometry; FIBTEM, fibrinogen thromboelastometry. Clotting time (CT, time from the start of measurement to the start of clotting, in seconds); alpha angle (tangent to the curve at 2 mm amplitude, in degrees, which reflects the rate of fibrin polymerization); maximum clot firmness (MCF, in mm, which reflects the maximum tensile strength of the clot); and lysis at 30 min (Ly30, in %; residual clot firmness 30 min after CT).
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Molecular diagnosis disclosed compound heterozygosity of the highly recurrent Spanish nonsense mutation p.G2177Wfs113* (c.6527insC) with a missense mutation p.G2434R (c.7300G>A) leading to the diagnosis of RDEB intermediate and consistent with reduced expression of C7 (Appendix 1 and Supplementary Figure 1). Ulcers in the oral mucosa, causing bleeding and unbearable pain, progressively worsened and failed to respond to a variety of repeated treatments (Figure 1A), causing frequent hospitalizations and emergency episodes. Table 1) were administered on a long-term basis, but barely improved gastric symptoms, pain, and pruritus. Therefore, in the context of compassionate use, a concomitant systemic cell-based treatment was offered in 2017 (Figure 1A). Common mild infusion-related adverse reactions were in remission within a few hours by the administration of ibuprofen and metoclopramide (Figure 1B). Hemostatic and ROTEM parameters such us platelet count (253 x 103/mul), coagulation time (11.5 seg), PT, aPTT, CT-EXTEM, MCF-EXTEM, and CT-INTEM were within the normal range and not affected by the infusion (Figure 1C).
Assessment of the therapeutic effect of systemically administered ADMSC (A) Photographic images of legs, abdomen and mouth before and at various time points after the treatment showing a cutaneous improvement clinimetrically quantified by two specific severity index/scores. (B) EBDASI and (C) BEBSS. Mucocutaneous improvement after the treatment parallels a decrease in the scores for (D) pain (VAS) and (E) itch (LIS), as well as an improvement in (F) patient's quality of life (QoL-5D). Red arrows indicate first, second and third infusion. *: esophageal dilation.
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Specifically, a progressive amelioration in the percentage of body surface covered by lesions dropped from 23 to 12% at 6 months after the first injection (Figure 2A). BEBSS and EBDASI, which measure changes in the mucocutaneous pathology, impressively reduced at 3 months, reaching minimal values at 6 and 9 months post-treatment, respectively (Figures 2B,C). On a VAS-scale, pain intensity decreased immediately after the third infusion reaching the minimum level at 9 months (Figure 2D). According to the LIS questionnaire, the patient experienced an improvement in all aspects of itch (except duration) 21 days after the first infusion, reaching minimum values after 6 months (Figure 2E). Regarding EuroQol-5D, the negative impact of the disease on patient's mobility, self-care and usual activity alongside maximum pain gradually improved, with the best QoL at 6 months after the treatment (Figure 2F). This population of likely immature neutrophils decreased gradually, disappearing completely 6 months post-treatment (Figure 4N) when the overall improvement in the disease was maximal (Figure 2) and the numbers and SSC of circulating granulocytes reached the lower values (Figure 4O).
Impact of systemically administered ADMSC on non-specific inflammatory markers. White blood cell count represented as percentage of (A) lymphocytes (B) neutrophils, and (C) eosinophils. Circulating levels of (D,E) positive and (F,G) negative acute phase reactants. Dashed lines represent the reference rank in age-matched general population. Circulating levels of (H) total TGF-beta and (I) MCP-1/CCL2 in the control group (Box plot) and the patient (black curve). Box plot representing the statistical median (white line), interquartiles (IQR; black box), and the lowest and highest data points (Tukey whiskers black lines) calculated from nine control individuals (three males and six females, aged from 24-63). The number of control individuals, after eliminating non-representative outliers, is indicated in every case (n). Red arrows indicate first, second, and third infusion.
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Low and high proportions of lymphocytes and neutrophils, respectively, were normalized after the first infusion; low eosinophil percentage reached normal values exclusively after the third infusion (Figures 3A-C). Amelioration in the mucocutaneous disease was accompanied by an improvement in nutritional and inflammatory markers (Supplementary Table 4 and Figures 3D-G), which were moderately abnormal at baseline. Decreased levels of prealbumin and 25-hydroxy-vitamin D, indicative of mild malnutrition, normalized after the second infusion for up to 6 months, along with a 3 kg weight gain (Figure 3G and Supplementary Tables 3, 4). In parallel, a substantial modulation in the patient's abnormal circulating levels of acute phase reactants occurred (Figure 3B). They were close to normal after 3 months and within control values 2 years after the first infusion, although transiently peaked at 1 year point when the patient had an episode of pharyngitis (Figure 3D). Increased levels of fibrinogen normalized after the second infusion for up to 2 years (Figure 3F). Circulating levels of serum cytokines (Supplementary Figure 3) were determined. Up-regulation of MCP-1/CCL2 after the first infusion was followed by a down-regulation of TGF-beta after the third infusion (Figures 3H,I) whilst IL10 levels were mostly steady (data not shown).
Impact of systemically administered ADMSC on peripheral blood populations. (A-I) Circulating lymphocyte populations. (A) Absolute numbers of lymphocytes (blood count tests). (B-I) Percentages of various subpopulations, within lymphocyte-gated cells, in patient and healthy control donors (CTL, mean +- SD; n = 6-7), analyzed by flow cytometry. (B) Percentages of B cells (CD19+) (C) Percentages of NK cells (CD56+CD3-, open circles, left axis) and CD56bright CD16- subset within total NK cells (filled diamonds, right axis). (D) Percentages of total (CD3+) T cells, (E) CD4 (CD4+CD3+), (G) CD8 (CD4-CD3+) T subpopulations and (H) TREG cells (CD25+Foxp3+) within CD4 T cells. Percentages of naive (CD45RA+CD45RO-) and memory (CD45RA-CD45RO+) cells within CD4 (F) and CD8 (I) T cell subpopulations. (J-O) Circulating myeloid populations. Absolute numbers of monocytes (J) and neutrophils (L) obtained in patient blood count tests. (K) Percentages of CD14+ cells within the myeloid gate (open circles, left axis) and CD16+ subset within CD14+ cells (filled diamonds, right axis) found in patient and healthy control donors (CTL, mean +- SD; n = 6), analyzed by flow cytometry. (M) Representative dot plots showing CD15 vs. CD33 expression on granulocyte (GR) and CD14+ gated cells of a control donor and patient samples as indicated. (N) Dot plots show forward (FSC) vs. size (SSC) scatter of granulocyte-gated population obtained by flow cytometry analysis. d, days after first infusion; mo, months after first infusion; yr, before (pre-treatment) and after (post-treatment) first infusion. (O) The line chart represents the SSC arithmetic means of granulocyte population in the patient and healthy control donors (CTL, mean +- SD; n = 7). Vertical grids indicate days of MSC infusion. Horizontal grids indicate ranges considered clinically normal.
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Decreased levels of prealbumin and 25-hydroxy-vitamin D, indicative of mild malnutrition, normalized after the second infusion for up to 6 months, along with a 3 kg weight gain (Figure 3G and Supplementary Tables 3, 4). ADMSC on the patient's PBL populations was monitored (Figure 4 and Supplementary Tables 7, 8). Absolute numbers of lymphocytes were low but within the reference range up to 6 months after treatment when a gradual increase took place (Figure 4A). The proportion of B cells was higher than the control range but changes after the treatment might not be clinically relevant (Figure 4B). ADMSC infusion (Figure 4D). The proportion of total CD3+ T cells, normal at all the time points in the study, progressively increased after the first infusion, largely because of the raise in the CD4+ T cell subpopulation (Figure 4E). Although MSC have been repeatedly described as potent inducers of immunotolerance, this effect was not reflected in an increase in FOXP3+CD25+ CD4 T cells in our patient (Figure 4H). Nevertheless, we observed an important transitory recovery of the markedly reduced proportion of memory T cells, which reached normal values after the third infusion up to 1 year (Figure 4F). Of note, CD8 memory cells doubled their proportion after ADMSC infusion to fourfold in absolute terms 1 year post-treatment (Figure 4I). The main myeloid populations were also analyzed. The absolute numbers of monocytes were low but within the reference range at baseline and stable after 6 months post-treatment (Figure 4J). From the first ADMSC infusion, this proportion decreased and was within the control range (Figure 4K). Interestingly, the remarkably low level of CD33 (Singlec3) expression in our patient's monocytes and granulocytes, an inhibitory receptor used as a typical myeloid marker whose involvement in disease is not yet completely understood was not modified after the treatment (Figure 4M). At baseline, the absolute number of neutrophils, although within the control range, was relatively high (Figure 4L). At this point, we also found an anomalous subpopulation with a larger size and cellular complexity (SSC) (Figure 4N) compatible with immature granulocytes. This population of likely immature neutrophils decreased gradually, disappearing completely 6 months post-treatment (Figure 4N) when the overall improvement in the disease was maximal (Figure 2) and the numbers and SSC of circulating granulocytes reached the lower values (Figure 4O).