Autologous Mononuclear Leukocytes Research Articles

Pathomorphological Features of The Cornea in Secondary Posttraumatic Dystrophy (Clinical Case)

Secondary corneal dystrophy is a group of diseases characterized by an inflammatory–dystrophic process in the cornea of the eye. This pathology is accompanied by a pronounced pain syndrome and leads to an irreversible decrease in vision, up to complete blindness. To date, many aspects of pathomorphological changes of the cornea in secondary dystrophies remain insufficiently studied due to the impossibility of conducting an intravital study of the cornea tissue. The article presents a case of morphological analysis of a tissue fragment of a pathologically altered cornea in secondary posttraumatic corneal dystrophy. Destructive changes in corneal tissue revealed during light microscopy indicate the presence of signs of fibrinoid swelling in the shell stroma with gross disorganization of the base substance and neovascularization. The study made it possible to correlate the data of pathomorphological changes and the clinical picture, as well as to evaluate the effectiveness of the chosen method of treatment of this pathology (intrastromal injection of a suspension of autologous mononuclear leukocytes of blood into the stroma of the damaged cornea).

Features of the local cytokine profile of patients with bullous keratopathy by using personalized therapy with cellular technologies

Background. Today we have active development of ophthalmic surgery, but the role of cytokines in the pathogenesis of bullous keratopathy of the cornea remains insufficiently studied at present.The aim. To study the dynamics of the local cytokine profile in bullous keratopathy by using personalized treatment with suspension of autologous blood mononuclears.Material and methods. Two groups of patients with bullous keratopathy were formed: the main group (30 people, 30 eyes), who received an intrastromal injection of a suspension of autologous blood mononuclears and comparison (28 people, 28 eyes), who received a course of pharmacotherapy. The level of interleukins (IL) – IL-10, IL-4, IL-6, IL-1ß, transforming growth factor beta-2 (TGF-β2), tumor necrosis factor alpha (TNF-α) was determined in the lacrimal fluid of the patient’s eye by enzyme immunoassay.Results. All patients before treatment showed an increase of IL-1ß in the lacrimal fluid by 3.3 times, IL-6 – by 4.2 times, TNF-α – by 2.0 times (p < 0.05); an increase in the level of IL-4, IL-10 and TGF-β2 by 1.1 times (p > ˂ 0.05). In the main group, after treatment, there was a decrease in IL-1ß by 2.0 times, IL-6 – by 2.1 times and TNF-α – by 1.8 times, and an increase in IL-10 by 1.5 times, IL-4 – by 1.9 times, TGF-β2 – by 1.4 times (p < 0.05), persisting for 12 months. There was a short-term decrease of IL-1ß by 1.7 times, IL-6 and TNF-α – by 1.2 times and an increase of IL-10, IL-4, TGF-β2 by 1.2 times (p >< 0.05) in the comparison group which then reached the initial values. Conclusion. There is an imbalance in the system of pro- and anti-inflammatory cytokines in bullous keratopathy in the lacrimal fluid. Injection of autologous mononuclear cells in the stroma of the cornea reduces the severity of the imbalance of the local cytokine system compared to the course of pharmacotherapy. Key words: bullous keratopathy, cellular technologies, autologous mononuclear leukocytes, cytokines>˂ 0.05), persisting for 12 months. There was a short-term decrease of IL-1ß by 1.7 times, IL-6 and TNF-α – by 1.2 times and an increase of IL-10, IL-4, TGF-β2 by 1.2 times (p ˂ 0.05) in the comparison group which then reached the initial values.Conclusion. There is an imbalance in the system of pro- and anti-inflammatory cytokines in bullous keratopathy in the lacrimal fluid. Injection of autologous mononuclear cells in the stroma of the cornea reduces the severity of the imbalance of the local cytokine system compared to the course of pharmacotherapy.

Clinical and Functional Results of the Use of Suspension of Autologous Mononuclear Leucocytes in the Surgical Treatment of Bullous Keratopathy

The purpose: to explore the clinical effectiveness of a new cell-based surgical method for the treatment of bullous keratopathy that includes intrastromal injection of autologous mononuclear leukocytes into the posterior third of the cornea stroma.Material and methods. The study included 58 patients with diagnosis of bullous kerathopathy. They were divided into 2 groups — the main group received surgical treatment by the presented method; and the comparison group received a course of conservative therapy.Results. In 10 days the patients in the main group had a normal structure of the cornea epithelium, slight edema of the stroma, thin folds of the Descemet’s membrane and moderate edema of the endothelium. Similar results in the comparison group were achieved in 1 month, and it was the best condition of the cornea. Later corneal edema increased. In 3 months the patients of the main group had a normal structure of corneal epithelium, single thin folds of the Descemet’s membrane, slight edema of the posterior third of the stroma only in the optical zone, and slight edema of the endothelium. The achieved results were preserved for 12 months. The best correct visual acuity (BCVA) in the main group was achieved by the 6th month. It increased 10 times from the initial level, and the corneal thickness decreased by 27 % from the initial level by the 9th month. In the comparison group, the BCVA was achieved by the 1st month. It increased 2.7 times from the initial level; the maximum decrease in the index of pachymetry by 6.7 % was achieved by the 3rd month from the initial level, and then these parameters increased.Conclusion. The using of a surgical method for the treatment of bullous keratopathy, including intrastromal injection of suspension of autologous mononuclear leukocytes, contributes to the relief of corneal syndrome, increases the transparency of the cornea and restores its normal thickness. These changes are accompanied by increase of BCVA in the postoperative period compared to the course of conservative therapy.

Рathomorphological features of corneal regeneration in experimentally induced dystrophy against the background of intrastromal administration of autologous mononuclear leukocytes

The purpose of research is study the features of the flow of reparative regeneration of the cornea in an experiment in vivo with induced bullous keratopathy after the introduction of a suspension of autologous mononuclear leukocytes in the posterior third of the corneal stroma. Materials and methods. The experiment was performed on 27 chinchilla rabbits (27 eyes). At the first stage of the experiment all animals were reproduced the model of corneal bullous keratopathy by mechanical damage and removal of its endothelial layer. At the second stage, 2 weeks after the development of the disease, the animals were divided, depending on the planned treatment, into 2 groups: the main, 12 animals once in the back third of the corneal stroma were administered a suspension of autologous mononuclear leukocytes; comparisons, 12 animals were treated by conservative therapy. Results. Reproduction of in vivo bullous keratopathy of the cornea by mechanical damage and removal of its endothelial layer is accompanied by rapid (within 2 weeks) the development of the disease with diffuse hydropic degeneration of the anterior epithelium, marked edema of the stroma with the bundle of collagen fibers. On the experimental model of the bullous keratopathy of cornea it was found that the introduction of a suspension of autologous mononuclear leukocytes in the posterior third of the corneal stroma in the treatment of the disease provides rapid (1.5 times) dehydration of the stroma, reduces the number of dystrophic cells in the anterior epithelium by 1.2 times compared with conservative treatment. The period of restoration of the normal thickness of the corneal slice in animals of the main group is reduced by 1.4 times. The conclusion. The introduction of autologous mononuclear leukocytes suspension into the posterior third of the corneal stroma in the treatment of experimental bullous keratopathy of cornea induces reparative regeneration, accelerating the restoration of damaged corneal structures and preventing its neovascularization.

Ectopic expression of HLA class II antigens on thyroid follicular cells: induction and transfer in vitro by autologous mononuclear leukocytes.

Ectopic expression of HLA class II antigens by thyroid follicular cells (TFC) might trigger the immune recognition of TFC-specific surface constituents by self-reactive T helper/inducer lymphocytes, with the consequent generation of organ-specific autoimmunity. To explore the alternative possibilities underlying this ectopic expression, i.e. that it either reflects a primary abnormality of the TFC or is secondarily induced by the autoimmune attack itself, we employed immunofluorescence staining of monolayers cultured from thyroid tissue of patients with autoimmune thyroid disease as well as neoplastic, nodular, and normal thyroid tissues to assess the role that autologous mononuclear leukocytes (MNC) and serum factors play in HLA class II induction and modulation in vitro. In all Graves' disease (GD) tissues that initially displayed HLA-DR (DR) antigens, this expression was transient. Primary TFC cultures from tissues with tumor-associated chronic thyroiditis (CT) showed more widespread and persistent DR expression than did cultures from GD tissues. However, in contrast with the findings in GD, there was no correlation between ectopic DR expression on TFC from the CT areas and the presence of organ-specific autoimmune markers. DR expression by TFC was detected in only one of the five nontoxic nodules studied, and involved a small proportion (approximately 20%) of the TFC. A similarly low proportion of DR-positive TFC was found in cultures from two papillary carcinomas, while much stronger DR expression was seen on TFC cultured from the contralateral lobes affected with CT. Only one of four follicular adenomas expressed DR on about 35% of the cultured TFC. In contrast with the rapid extinction of DR expression on TFC cultured from GD tissues after depletion of infiltrated MNC, DR antigen loss did not occur or was significantly delayed when parallel TFC monolayers were cocultured in the presence of autologous MNC from the intrathyroidal infiltrates. Coculture of DR-negative TFC from normal tissues with autologous peripheral blood MNC in individuals without detectable thyroid autosensitization also resulted in pronounced induction of DR expression on the TFC. Purified T lymphocytes from the same peripheral blood MNC preparations, however, did not induce DR expression on these normal TFC. Similarly, MNC obtained from two follicular adenomas, mostly macrophages, did not induce DR expression when cultured with autologous TFC from the same lesions. Addition of 15% autologous serum did not prevent progressive extinction of DR expression on TFC cultured from GD or tumor-associated CT tissues after depletion of infiltrated MNC.(ABSTRACT TRUNCATED AT 400 WORDS)

Adoptive immunotherapy for stage IV renal cell carcinoma: A novel protocol utilizing periodate and interleukin-2-activated autologous leukocytes and continuous infusions of low-dose interleukin-2

In a pilot study involving 13 patients with advanced stage IV renal cell carcinoma, anti-tumor effects and toxicity of a novel form of adoptive immunotherapy were determined. The protocol utilizes infusions of autologous mononuclear leukocytes treated with the oxidizing mitogen sodium periodate (IO 4 −) and cultured in medium containing human recombinant interleukin-2 (IL-2), and continuous infusions of low-dose IL-2 (mean ± SD dose = 39.5 ± 8.6 × 10 3 U/kg/24 hours). Leukocytes (5 to 10 × 10 9) were removed by leukapheresis three times per week, mononuclear cells were separated, activated with IO 4 − and cultured in medium containing IL-2 (500 U/ml) for 48 to 72 hours. The cells were re-infused following the next leukapheresis procedure. IL-2 was administered five days per week. Treatment was continued for two three-week cycles. An increase in peripheral blood mononuclear cells bearing the natural killer cell (NK) surface marker, Leu 11, an increase in NK- and antibody-dependent cell-mediated cytotoxicity, and a slight increase in spontaneous cytotoxicity for non-NK targets were noted. Regressions (more than 50 percent decrease in tumor mass) of pulmonary, liver, bone, or soft tissue metastases were induced in six patients. Severe fluid retention did not develop in any patient and no patient required treatment in the intensive care unit. Five of the patients who showed a response have experienced a relapse at 5.2 ± 1.0 (mean ± SD) months. These observations indicate that IO 4 −/IL-2-activated killer cells plus continuous infusions of low-dose IL-2 can result in regression of metastatic renal cell carcinoma.

Stimulation of T cells by autologous mononuclear leukocytes and epidermal cells in psoriasis

Based on reports suggesting aberrant cell-mediated immunity and altered infiltration of immunocompetent cells into the skin in psoriasis, we studied the stimulation of T cells by autologous non-T mononuclear leukocytes (autologous mixed lymphocyte reaction, AMLR) and by epidermal cells isolated from lesional and clinically uninvolved skin in psoriasis (autologous mixed epidermal cell lymphocyte reaction, AMECLR). Age- and sex-matched individuals served as controls. We found that the AMLR in psoriasis (n = 11) was similar to that in healthy controls (n = 16); furthermore, cell proliferation was alike in the presence of either 5% AB-serum or autologous serum. By contrast, while the AMECLR in healthy controls (n = 9) resembled that in psoriatics employing epidermal cells from univolved skin, epidermal cells from lesional sites (n = 10) induced a significantly higher proliferation of autologous T cells in the AMECLR (P less than 0.01). We conclude that the in vitro stimulation of T cells by non-T mononuclear leukocytes is normal in psoriasis and is not regulated by autologous serum. Lesional psoriatic epidermal cells, however, are more active in stimulating autologous T cell proliferation than cells from univolved psoriatic or normal epidermis.

Antigen presentation by Hodgkin's disease cells.

The L428 tumor cell line is a long-term tissue culture of Reed-Sternberg cells which was derived from the pleural effusion of a patient with Hodgkin's disease. The L428 cells express all known cell surface antigens, cytochemical staining, and cytologic features of freshly explanted Reed-Sternberg cells. In addition to the previously described HLA-DR cell surface antigens, the L428 cells are now demonstrated to express both DS and SB alloantigens. Thus, the L428 cells express all of the known subclasses of the human immune response genes that are located in the major histocompatibility complex. Furthermore, the L428 cells are capable of presenting soluble antigen to T cells in a genetically restricted fashion. T cell lines were established from normal donors previously immunized with tetanus toxoid. The T cells utilized were incapable of tetanus toxoid-induced proliferation unless antigen-presenting cells were added to the cultures. However, T cells from the two normal donors, which like the L428 cells expressed HLA-DR 5, demonstrated significant proliferative responses when cultured with tetanus toxoid and L428 cells. No proliferative response was observed when the L428 cells were used as antigen-presenting cells for a DR (4,-), DR (2,-) or DR (1,7) T cell line. The tetanus toxoid dose-response curve was similar regardless of whether autologous mononuclear leukocytes or L428 cells were used as antigen-presenting cells. The T cell proliferation induced by soluble antigen was also blocked by anti-HLA-DR antibody. Thus, functionally, Hodgkin's disease may be classified as a tumor of antigen-presenting cells.

Lytic effects of serum and mononuclear leukocytes on oral epithelial cells in recurrent aphthous stomatitis

A radioisotope-release assay, utilizing 51Cr-labeled epithelial cells derived from non-keratinizing oral mucosa, was developed to investigate in vitro cytolytic reactions correlating with recurrent aphthous stomatitis (RAS). The cytolytic effects of sera and mononuclear leukocytes from patients in the early stage of ulceration were compared with those from matched RAS-negative control subjects. RAS sera induced significantly more cytolysis than did matched control sera. Heating the RAS or control sera for 30 min at 56°C abrogated their cytotoxic activity. RAS mononuclear leukocytes, like their matched controls, showed no significant direct cytotoxicity. Heat-inactivated RAS or control sera acting in concert with RAS or control mononuclear leukocytes showed no consistent cytolytic effects. However, the heat-inactivated sera of some RAS patients, when combined with autologous mononuclear leukocytes, induced significantly more cytolysis than did either component acting alone. Thus heat-labile humoral factors and, in some cases, mononuclear leukocytes acting in concert with heat-stable serum factors are implicated in RAS-associated in vitro cytolytic reactions. These findings suggest that the effector mechanisms of such reactions include both complement-mediated and antibody-dependent cell-mediated cytotoxicity.

Role of monocytes, alloantigens, and individual reactivity in the response of human lymphocytes to mitogenic factor.

The activity of mitogenic factors generated in human mixed lymphocyte cultures (MLC-MF) was analyzed with unstimulated mononuclear leukocytes used as responder cells. Unseparated or adherent, but not nonadherent, irradiated, autologous mononuclear leukocytes were found to significantly enhance the mitogenic activity of MLC-MF. The effect of MLC-MF was not inhibited by hydrocortisone at concentrations that inhibited MLC-MF production, indicating that the MLC-MF effect is direct and is not mediated through further production of mitogenic factors. Experiments in which MLC-MF was produced in a MLC between a HLA-D homozygous individual and her child suggested that soluble alloantigens do not play a role in the MLC-MF effect. Studies in one individual with MLC responses higher than other individuals indicated average MLC-MF production but above average response to MLC-MF. We conclude that MLC-MF is directly mitogenic for unstimulated lymphocytes, without requiring the simultaneously presence of either mitogen or antigen. In addition, the proliferative MLC reaction of antigen. In addition, the proliferative MLC reaction of individuals with strong responses to MLC-MF may be influenced significantly by the nonspecific clones recruited by MLC-MF.

Induction of autoantibody-producing cells after the coculture of haptenated and normal human mononuclear leukocytes.

The coculture of normal human peripheral blood mononuclear leukocytes (PBL) and autologous mononuclear leukocytes coupled to the trinitrophenyl (TNP) hapten (TNP-PBL) was found to induce a polyclonal activation of antibody-producing cells. The polyclonal activation of antibody-producing cells was demonstrated by detecting the induction of cells producing antibody to sheep red blood cells using a complement-dependent, direct, hemolytic plaque-forming cell (PFC) assay. A ratio of four normal to one haptenated mononuclear leukocyte was found to be optimal for inducing the polyclonal activation of antibody-producing cell in these cultures. The plaque-forming cells assay in these experiments utilized monolayers of indicator red cells. Further evidence for the polyclonal induction of antibody-producing cells by TNP-PBL was provided by demonstrating PFC on monolayers of not only sheep red blood cells, but also autologous human red cells, bromelain-treated autologous red cells, TNP-coupled human and sheep red cells, and human autologous red cells coupled to human heat-aggregated IgG with chromic chloride. Thus cells secreting antibody to TNP, human red cells, and human IgG were induced. Anti-IgG and anti-human red cell-producing cells were first detected on Day 2 of culture and were still present on Day 9. Mononuclear leukocytes altered by chemical haptenation polyclonally stimulate normal mononuclear leukocytes to become antibody-producing cells. This polyclonal stimulation of antibody-producing cells includes cells producing antibodies to human IgG and human autologous red blood cells suggesting that autoantibody-producing cells are induced.