Combined Use of Tocilizumab and Mesenchymal Stromal Cells in the Treatment of Severe Covid-19: Case Report

Alexandra Cristina Senegaglia, Carmen Lúcia Kuniyoshi Rebelatto, Claudio Luciano Franck, Juliana Souza Lima, Lidiane Maria Boldrini-Leite, Debora Regina Daga, Cleverson Alex Leitão, Patrícia Shigunov, Ana Paula de Azambuja, Elisa Bana, Daniela Boscaro Marsaro, Bruna Schaidt, Andressa Micosky, Valderez Ravaglio Jamur, Yara Schluga, Isadora May Vaz, Lisandro Lima Ribeiro, Alejandro Correa, E Paulo Roberto Slud Brofman, Alexandra Cristina Senegaglia, Carmen Lúcia Kuniyoshi Rebelatto, Claudio Luciano Franck, Juliana Souza Lima, Lidiane Maria Boldrini-Leite, Debora Regina Daga, Cleverson Alex Leitão, Patrícia Shigunov, Ana Paula de Azambuja, Elisa Bana, Daniela Boscaro Marsaro, Bruna Schaidt, Andressa Micosky, Valderez Ravaglio Jamur, Yara Schluga, Isadora May Vaz, Lisandro Lima Ribeiro, Alejandro Correa, E Paulo Roberto Slud Brofman

Abstract

The coronavirus pandemic is one of the most significant public health events in recent history. Currently, no specific treatment is available. Some drugs and cell-based therapy have been tested as alternatives to decrease the disease's symptoms, length of hospital stay, and mortality. We reported the case of a patient with a severe manifestation of COVID-19 in critical condition who did not respond to the standard procedures used, including six liters of O2 supplementation under a nasal catheter and treatment with dexamethasone and enoxaparin in prophylactic dose. The patient was treated with tocilizumab and an advanced therapy product based on umbilical cord-derived mesenchymal stromal cells (UC-MSC). The combination of tocilizumab and UC-MSC proved to be safe, with no adverse effects, and the results of this case report prove to be a promising alternative in the treatment of patients with severe acute respiratory syndrome due to SARS-CoV-2.

Keywords: COVID-19; mesenchymal stromal cells; tocilizumab; umbilical cord.

Conflict of interest statement

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Fig. 1.
Fig. 1.
Study design. (A) Umbilical cord-derived mesenchymal stromal cells (UC-MSC) were isolated, expanded, and evaluated for surface markers, viability, presence of fungi and bacteria, and chromosomal abnormalities. The patient received three infusions of 500.00 UC-MSCs/kg and two infusions of 400 mg of tocilizumab. The patient’s clinical and laboratory evaluations were performed pre-MSC infusion (D1) and on days 2, 4, 6, 14, 60, and 120. (B) Schematic representation of the days before (minus) and after the first infusion of UC-MSC.
Fig. 2.
Fig. 2.
Quality control of umbilical cord-derived mesenchymal stromal cells (UC-MSC). (A) Cell characterization; (B) Cell viability. (C) Cytogenetics test. The cells showed a high expression of CD29, CD73, CD90, and low expression of CD14, CD19, CD34, CD45, and HLA-DR markers (A). Cell viability analysis showed less than 8% of dead cells (7-AAD) and less than 1% of cells in apoptosis (Annexin-V) (B). The UC-MSC did not show any chromosomal aberrations. Ann: Annexin; 7-AAD: 7-amino actinomycin.
Fig. 3.
Fig. 3.
Relative viral quantification. (A) The viral RrRP gene’s presence was normalized by the human RNApol gene’s presence and decreased gradually from the viral gene in the patient’s samples from 1 (D1) to 0.06 (D6) undetectable in D14. (B) The viral slope (-0.9422) and R2 (0.9871) was established after linearization of the data by natural logarithm (ln(x)) and obtaining the linear equation.
Fig. 4.
Fig. 4.
Immunophenotypic profile. (A) An increase in the absolute number of T lymphocytes/ µL also has been observed to have progressively increased from 148.6 (D0), 642.6 (D6), 607.4 (D14), 485,7(D60), and 775.4 (D120); CD4 T lymphocytes, 102 (D0), 481.2 (D6), 459.5 (D14), 358.0 (D60), and 567.9 (D120) and Treg lymphocytes 10.8 (D0), 34 (D6), 29.8 (D14), 25.9 (D60), and 14.2 (D120). Plasmablasts, in contrast, progressively decreased from 52 (D0) to 0.01 (D120). (B) Lymphocytes gated on CD4 (red/orange) and analyzed in merge form of the sequencing days (D0, D1, D2, D4, D6, and D14), showing the decrease in CD25+/CD127neg T cells—TREG (Orange scale). (C) B-cell lymphocytes gated on CD19 (red) and Plasmablasts gated on CD19+CD38++ (blue scale), showing a decrease in plasmablasts number from D0 to D14. TL (T Lymphocyte); CD4 (cluster of differentiation); TREG (T regulatory cells); BL (B Lymphocyte).
Fig. 5.
Fig. 5.
Chest computed tomography scan. (A) Chest CT scan without contrast, the axial section at the lower lobes level. Pre-transplantation, showing ground-glass opacities associated with crosslinking (mosaic paving) and subpleural lines, predominantly peripheral and basal. D14 shows an increase in the extent of ground-glass opacities, now with a more significant amount of peribronchovascular opacities. D60 shows marked complete regression of the opacities previously described. D120 shows almost complete regression of the opacities previously described. (B) Each lobe was assigned a score that was based on the following: score 0, 0% involvement; score 1, less than 5% involvement; score 2, 5% to 25% involvement; score 3, 26% to 49% involvement; score 4, 50% to 75% involvement; and score 5, greater than 75% involvement. Scores of 0 to 5 were determined for each lobe, with a total possible score of 25 (adapted from Li et al.).

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