Kathleen R. Mueller

Thymosin-α1 stimulates maturation of CD34+ stem cells into CD3+4+ cells in an in vitro thymic epithelia organ coculture model

The effect of thymosin-alpha1 on thymopoiesis is largely unknown. Thymosin is found in the cortical and medullary thymic epithelia, as well as in nurse cells; thus, it is hypothesized that thymosin may affect both early and late stage of thymocyte maturation. In this study, the effect of thymosin-alpha1 on thymopoiesis was determined by coculturing in vitro CD34+ stem cells (SC) with allogeneic cultured thymic epithelia fragments (CTEF) for 1-4 weeks and analyzing T-cell maturation by flow cytometry. Thymosin-alpha1 significantly enhanced the cell number (e.g., proliferation) of mononuclear cells obtained at 2 and 4 weeks of the SC-CTEF cocultures (P < 0.01 and < 0.05, respectively). In particular, thymosin-alpha1 stimulated expression of CD3+ cells at 3 and 4 weeks (P < 0.05). The predominant subpopulation increased by thymosin stimulation was single positive mature CD4+ cells, which was confirmed to occur within the SC-CTEF thymic organ tissue by laser confocal immunofluorescence microscopy. Thymosin stimulation tended to enhance IL-7 synthesis, critical cytokine in the maturation of thymocytes. In summary, this is the first study to demonstrate that thymosin-alpha1 enhanced thymopoiesis of CD34+ stem cells in humans using an in vitro model of differentiation using stem cells and cultured thymic epithelial fragments cocultures. Furthermore, the thymosin significantly increased expression of CD3+4+ T cells.

Serum anti-Aspergillus fumigatus antibodies by immunoblot and ELISA in cystic fibrosis with allergic bronchopulmonary aspergillosis☆

Allergic bronchopulmonary aspergillosis (ABPA) occurs with a prevalence of 5% to 15% in patients with cystic fibrosis (CF). Because of the frequent colonization with Aspergillus fumigatus (Af) in CF, the causative agent of ABPA, antibody reactivity to Af proteins is frequently observed, which obscures the diagnosis of ABPA. Patients with CF are also categorized according to the presence of positive skin test responses to Af and/or the presence of positive precipitins. In this study we used ELISA and immunoblot assay to detect IgE and IgG anti-Af antibodies in patients with CF and ABPA (n = 13) compared with other groups of patients with CF: those with positive skin test and positive precipitin results (n = 18), those with positive skin test and negative precipitin results (n = 14), those with negative skin test and positive precipitin results (n = 10), and those with negative skin test and negative precipitin results (n = 35). IgE and IgG anti-Af antibodies were significantly elevated in patients with ABPA as determined by both immunoblot assay (p < 0.01) and ELISA (p < 0.01). However, detection of Af antibodies by ELISA was more sensitive in discriminating patients with CF and ABPA from patients with CF who had positive skin test and positive precipitin results but lacked radiographic and clinical evidence of ABPA. In patients with CF and ABPA the immunoblot assays demonstrated a multitude of IgE, IgG, and IgA antibody responses to Af proteins, which ranged in molecular weight from 14 kd to greater than 106 kd. The level of IgE anti-Af antibody to individual proteins decreased during remissions of ABPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of Thymopoiesis of CD34+ Cell Maturation by HIV‐1 in an In Vitro CD34+ Cell and Thymic Epithelial Organ Culture Model

The mechanisms by which HIV‐1 affects thymopoiesis were determined by preincubating CD34+ cells or cultured thymic epithelial (CTE) cells with lymphotropic (T‐) and monotropic (M‐) strains of HIV‐1 in an in vitro CTE organ and CD34+ cell coculture model that allows for analysis of development of thymocytes and mature T cells.

Abnormal in vitro thymocyte differentiation in a patient with severe combined immunodeficiency-Nezelof`s syndrome

Anin vitro coculture model system of CD34+ stem cells and allogenic cultured thymic epithelia fragments was used to evaluate thymocyte differentiation in a 9-month-old child of Amish descent with Nezelof syndrome. Though the patients stem cells differentiate to acquire normal expression of CD2 and CD7, later steps of maturation were abnormal. There was detectable but reduced expression of CD3 and CD4 phenotypes. CD44+ expression, however, was markedly reduced. CD44 is an adhesion molecule, interacting with the matrix ligands hyaluronan and fibronectin, and is expressed early in thymocyte differentiation and subsequently in mature T cells. It is hypothesized that abnormal expression of CD44 in a variant of severe combined immunodeficiency, Nezelofs syndrome, interferes with normal thymocyte and thymic epithelial interaction, which leads to abnormal thymocyte differentiation.

T Cell Cytotoxicity in Cystic Fibrosis: Relationship to Pulmonary Status

T cell cytotoxicity (CTL) to an allogeneic lymphocyte target was evaluated in patients with cystic fibrosis (CF) before and during pulmonary exacerbations (group 1) compared to another group of CF patients who had stable pulmonary disease activity during their two study periods (group 2). CTL activity was significantly decreased in group 1 subjects studied prior to their pulmonary flares and in group 2 CF patients compared to normal controls at effector:target ratios of 12.5:1 and 6.25:1 (p less than 0.05 and p less than 0.05, respectively). Furthermore, in group 1, CTL lysis was significantly decreased during pulmonary flares compared to before flares at the 25:1 and 12.5:1, effector:target, (p less than 0.05 and p less than 0.05, respectively). T suppressor cell activity as measured by effect on in vitro control B cell IgM synthesis was significantly increased during pulmonary flares (p less than 0.05). Diminished CTL may be partially responsible for persistent colonization of Pseudomonas aeruginosa in CF.

Development of a Method of Thymocyte Differentiation of Bone Marrow-Enriched CD34+CD38− Cells in Postnatal Allogeneic Cultured Thymic Epithelia to Evaluate Immunodeficiency Disorders

An in vitro model of CD34+CD38− stem cell (SC) differentiation in postnatal cultured thymic epithelia fragment (CTEF) cocultures is described. Sequential phenotypic analysis of the progeny of the SC‐CTEF demonstrated predominantly thymocytes and minor populations of promyelocytes, monocytes and natural killer cells. Triple‐positive CD3+CD4+CD8+, double‐positive CD4+CD8+, and mature single‐positive CD4+ and CD8+ T cells, which were TCRαβ+, were identified indicating normal thymocyte maturation. In kinetic studies, mature single‐positive CD4+ T cells increased from 29% of total cells at one week to 54% at four weeks of coculture. These findings demonstrate that coculture of bone marrow‐derived SC and allogeneic cultured thymic epithelia in vitro results in continuous normal predominantly thymocyte differentiation. The SC‐CTEF cocultures were then infected with two different strains of human immunodeficiency virus. CD4+ thymocytes were markedly decreased. However, inhibition of early thymocyte maturation steps was also suggested by the presence of increased triple‐negative and CD44+CD25−CD3− thymocytes and decreased CD44+CD25+ thymocytes. This model system of thymocyte maturation will be useful in the evaluation of primary T cell immunodeficiency disorders, gene therapy of SC and pharmacological augmentation of thymic function.

Suppression of Mixed Lymphocyte Culture by Uremic Adherent Spleen Cells and Peritoneal Macrophages Is Dependent on a Cyclophosphamide-Sensitive Cell

A model of experimentally induced uremia in the rat has been used to study the effect of uremia on the response of spleen cells to alloantigens. The proliferative ability of uremic spleen cells in mixed lymphocyte culture is significantly suppressed when compared to that of cells from control animals. This suppression appears to be due to both adherent suppressor cells which can be eliminated by adherence to rayon wool and to the inability of uremic T cells to respond to alloantigens. In addition, unstimulated peritoneal macrophages ( PMO ) obtained from uremic rats were also shown to be suppressive to the response of control spleen cells to alloantigens. The suppression by uremic adherent spleen cells and PMO is regulated by cyclophosphamide-sensitive cells.