AMNIOTIC MEMBRANE TRANSPLANTATION AT THE DEPARTMENT OF OPHTHALMOLOGY OF THE UNIVERSITY HOSPITAL BRNO.

BACKGROUND: The aim of the work is a discussion of amniotic membrane transplantation at the Eye Clinic of the University Hospital Brno and a retrospective evaluation of a group of patients for the period 2014-2019 who were treated for various indications. METHODS: Retrospective evaluation of the number and effectiveness of individual types of amniotic membrane in a group of patients after amniotic membrane transplantation (AMT) for various indications. A total of 134 patients were included in the study group, of which 68 were men and 66 were women. The median age was 70 years. The total number of amniotic membrane transplants performed during the selected six years was 139, with half the distribution using frozen (69 eyes) and lyophilized amniotic membrane (70 eyes). The AMT technique was chosen based on the initial finding and diagnosis. The type of amniotic membrane used (lyophilized vs. frozen) depended on the urgency of the procedure. RESULTS: The number of amniotic membrane transplantations was evaluated in a group of 134 patients (139 eyes) and their effectiveness in individual diagnoses was demonstrated. During the follow-up period, more transplantations of amniotic membranes were performed during hospitalization than in the outpatient department, both types of membranes (frozen and lyophilized). Amniotic membrane transplantation during hospitalization was performed in 89 eyes, in the outpatient mode in 50 eyes. Indications for amniotic membrane transplantation included microperforation and corneal perforation (30 eyes), non-healing corneal defects (21 eyes), descemetocele (19 eyes), neurotrophic defects (16 eyes), ablation of pterygium (12 eyes) and corneal lysis (11 eyes). eyes). Other conditions (conjunctival lesions, fornix reconstruction, burns, peripheral ulcerative keratitis, ocular cicatricial pemphigoid and keratectomy) were represented in smaller numbers. Despite the very diverse group of indications and the advanced age of the patients, a very good efficacy of the performed amniotic membrane transplantations was found. Some patients died during the follow-up period, so the limitation of work is short and unequal follow-up period. CONCLUSION: The success of the procedure depends not only on the correct timing, indication and technique of transplantation, but also on patient compliance and well-functioning cooperation of regional ophthalmologists.

[Selective depletion of alloreactive T cells and study of anti-tumor activity of specific T cell clones in patients with leukemia]. / Selektivní deplece aloreaktivních T lymfocytu a studium protinádorové aktivity specifických klonu T lymfocytu u pacientu s leuikémií.

BACKGROUND: Graft-versus-host disease (GVHD) is a severe complication of allogeneic transplantation of hematopoietic stem cells. Donor T cells play a major role in GVHD leading to the host tissue damage, mainly the skin, liver, and gastrointestinal tract. A selective depletion using an anti-CD25 immunotoxin can eliminate harmful alloreactive T cells while preserving other donor T cells with antileukemic and antiinfectious reactivity. PATIENTS AND METHODS: We performed 15 mixed lymphocyte reactions with clinical specimens from 12 patients with various types of leukemia (7x AML, 3x ALL, 1x CML, 1x CLL) and PBMC from 15 healthy volunteers from Transfusive station FN Brno Bohunice. RESULTS: In our experiments we have demonstrated, that antileukemic (GVL) effect of donor, especially CD4+ T cells was well preserved (7.46%), while unfavourable alloreactive (GVH) reaction of donor T cells was completely removed. The graft-versus-host (GVH) reactivation of donor cells was negligible ever after repeated stimulation with irradiated patient's PBMC. CONCLUSION: We have shown that anti-CD25 immunotoxin (IT), RFT5-SMPT-dgA, launched against alpha chain for human interleukin 2 (IL-2), led to long-term selective depletion of alloreactive donor T cell clones while their antileukemic activity was well preserved. Base on our results the clinical phase I/II study was designed. This study was initiated in year 2007 in three clinical centers in Czech Republic.

Cell surface detection of HLA-E gene products with a specific monoclonal antibody.

An HLA-E-specific monoclonal antibody (mAb) was obtained by immunization of human beta2-microglobulin transgenic mice (M-TGM) with spleen cells from double transgenic mice expressing HLA-E molecules (EM-TGM). This mAb, designated V16, specifically recognizes in flow cytometry analysis the HLA-E expressing mouse cells, whereas it does not bind to mouse cells expressing various HLA class I molecules (HLA-A2, -A3, -A11, -A26, A29, -B7, -B27, -Cw3, -Cw7, and HLA-G). V16 mAb binds efficiently to human EBV-infected B lymphocytes, PHA blasts and PBL, thus establishing the surface expression of HLA-E in vivo on these cells.

In vitro activation of CMV-specific human CD8(+) T cells by adenylate cyclase toxoids delivering pp65 epitopes.

Human CMV infects between 50-85% of healthy individuals and can cause live-threatening infections in immunocompromised patients. Therefore, peptide vaccination is being developed as a promising immunotherapeutic approach for treatment of patients at risk of CMV disease. The enzymatically inactive toxoid of Bordetella adenylate cyclase (CyaA-AC(-)) was shown to be an efficient tool for delivery of peptide epitopes and stimulation of Ag-specific T-cell immune responses. We investigated here the capacity of two CyaA-AC(-) constructs to deliver epitopes derived from the CMV phosphoprotein pp65 for activation of human T cells in vitro. Expansion of γ-IFN-secreting CMV-specific CD8(+) T cells, as well as increase of total IFN-γ and TNF-α production by PBMCs from CMV-seropositive donors were observed after in vitro stimulation with CyaA-AC(-) constructs carrying CMV epitopes, whereas limited activation of immune response occurred with free peptides. The activation of immune response was confirmed by expansion of CMV-specific T-cell clones and anti-CMV cytotoxic effect of stimulated PBMCs. These data open the way to clinical evaluation of CyaA-AC(-) constructs as tools for detection and expansion of CMV-specific T-cell immune responses for diagnostic and immunotherapeutic applications against CMV-associated diseases.

Cutting edge: requirement of class I signal sequence-derived peptides for HLA-E recognition by a mouse cytotoxic T cell clone.

The human nonclassical MHC class I molecule HLA-E has recently been shown to act as a major ligand for NK cell inhibitory receptors. Using HLA-E-expressing transgenic mice, we produced a cytotoxic T cell clone that specifically recognizes the HLA-E molecule. We report here that this T cell clone lyses HLA-E-transfected RMA-S target cells sensitized with synthetic class I signal sequence nonamers. Moreover, this T cell clone lyses human EBV-infected B lymphocytes, PHA blasts, and PBL, formally demonstrating the surface expression of HLA-E/class I signal-derived peptide complex on human cells. Furthermore, these data show that HLA-E complexed with class I signal sequence-derived peptides is not only a ligand for NK cell inhibitory receptors, but can also trigger cytotoxic T cells (CTL).

Cell-surface expression and alloantigenic function of a human nonclassical class I molecule (HLA-E) in transgenic mice.

We have introduced the gene (E*01033) encoding the heavy chain of the human nonclassical MHC class I Ag, HLA-E, into the mouse genome. Two founder mice carry a 21-kb fragment, the others bear an 8-kb fragment. Each of the founder mice was mated to mice of an already established C57BL/10 transgenic line expressing human beta2-microglobulin (beta2m). Cell surface HLA-E was detected on lymph node cells by flow cytometry only in the presence of endogenous human beta2m. However, HLA-E-reactive mouse CTL (H-2-unrestricted) lysed efficiently the target cells originating from HLA-E transgenic mice without human beta2m, showing that the HLA-E protein can be transported to the cell surface in the absence of human beta2m, presumably by association with murine beta2m. Rejection of skin grafts from HLA-E transgenic mice demonstrates that HLA-E behaves as a transplantation Ag in mice. HLA-E transgenic spleen cells are effective in stimulating an allogeneic CTL response in normal and human classical class I (HLA-B27) transgenic mice. Furthermore, results from split-well analysis indicate that the majority of the primary in vivo-induced CTL recognizes HLA-E as an intact molecule (H-2-unrestricted recognition) and not as an HLA-E-derived peptide presented by a mouse MHC molecule, although a small fraction (ranging from 4 to 21%) of the primary in vivo-induced CTL is able to recognize HLA-E in an H-2-restricted manner. Based on these observations, we conclude that HLA-E exhibits alloantigenic properties that are indistinguishable from classical HLA class I molecules when expressed in transgenic mice.