Anna Pituch-Noworolska

Isotype-specific regulation of MHC class II gene expression in human monocytes by exogenous and endogenous tumor necrosis factor

The control of expression of MHC class II molecules on antigen-presenting cells is important for the induction of immunity, while aberrant expression of these molecules plays a role in the immunopathology of autoimmune diseases. This study explored the role of tumor necrosis factor alpha (TNF) in controlling the level of HLA class II mRNA in human monocytes. Exposure of monocytes to exogenous recombinant TNF (rTNF) selectively up-regulated DRα-mRNA but not DP or DQα-mRNA. Inhibitors of TNF synthesis, pentoxifylline (PTX) and thalidomide, inhibited TNF mRNA accumulation in LPS-activated monocytes and down-regulated DR mRNA but not DP or DQ mRNA. The inhibitory effect of anti-TNF monoclonal antibody (MAb) indicated that endogenously generated TNF acted extracellularly. Anti-p75 TNF-R2 receptor and to a lesser extent anti-p55 TNF-R1 MAbs inhibited TNF-mediated up-regulation of DR mRNA and TNF mRNA. Taken together, this implies that endogenously generated TNF plays a role in controlling isotype-specific MHC class II gene expression in human monocytes/macrophages. These results may have some implications for anti-tumor response and autoimmunity.

The regulation of polyclonal immunoglobulin synthesis by FcR+ and FcR− monocyte subsets

FcR+ and FcR- monocyte subsets were added to the pokeweed mitogen (PWM) or Staphylococcus aureus Cowan I-stimulated cultures of peripheral blood mononuclear cells (PBMC) or to PBMC depleted of monocytes. The numbers of immunoglobulin-secreting cells (ISC) and cells with intracytoplasmic immunoglobulins (PC) were evaluated 6 days later. The addition of FcR- subset increased the number of ISC in cultures of PBMC stimulated with PWM and reconstituted the response of monocyte depleted PBMC. In contrast, FcR+ monocytes suppressed PWM-induced response and, when added in high dose, also that induced by S. aureus. The FcR+ monocytes suppressed the response by inhibition of immunoglobulin secretion but not the development of PC. This suggests that FcR+ monocytes may modulate humoral response by preferential inhibition of the final differentiation of B lymphocytes into ISC.

Pokeweed mitogen activated suppressor T-cells preferentially reduce immunoglobulin secretion by differentiated B-lymphocytes

In contrast to pokeweed mitogen (PWM), S. aureus Cowan I (SAC) does not activate suppressor T-cells in cultures of human peripheral blood mononuclear cells, although the SAC induced response leading to the appearance of immunoglobulin secreting cells (ISC) is suppressed by PWM activated suppressor T-cells. Therefore, cultures co-stimulated by SAC and PWM were chosen to find out which stage of the SAC triggered B-cell response is controlled by PWM activated suppressor T-cells. By incorporation of [3H]thymidine and determination of B-cell number in culture it was shown that neither PWM itself nor PWM induced suppressor T-cells interfere with SAC induced B-cell proliferation. The final stages of B-cell maturation were monitored by parallel evaluation of cells producing immunoglobulins (cells with intracytoplasmic immunoglobulins) and secreting them (plaque assay). It was shown that PWM activated suppressor T-cells reduce the number of ISC more effectively than the number of immunoglobulin producing cells, indicating that these two features of differentiated B-lymphocytes may be independently controlled.

THE PHOTODYNAMIC EFFECT OF VICTORIA BLUE BO ON PERIPHERAL BLOOD MONONUCLEAR AND LEUKEMIC CELLS

Abstract— The photodynamic effect of Victoria blue BO (VB‐BO) and photoirradiation on peripheral blood mononuclear cells was studied. The cells were preincubated with VB‐BO followed by photoirradiation and overnight culture. The highest percentage of dead cells (propidium iodide assay in flow cytometry) was seen in the monocyte population. The lymphocytes showed a lower sensitivity to VB‐BO photodynamic action than the monocytes (12%vs 80% of Pi‐positive cells). The effect of VB‐BO and phototreatment on lymphocyte function was studied using a mitogen‐induced proliferation assay. A decrease of mitogen response was observed. The VB‐BO and photoirradiation were also used on leukemic cells. The leukemic cells from acute myeloid leukemia and B precursors leukemia were sensitive to VB‐BO photodynamic action. The high VB‐BO sensitivity of monocytes and leukemic cells (myeloid and lymphoid B derived) suggests possible application of VB‐BO for selective depletion of monocytes or sensitive leukemic cells.

Antistriational antibodies during Toxocara canis, Trichinella spiralis infections

The parasitic infections (Toxocara can is, Trichinella spiralis) are characterized by general and local symptoms including fever, muscle pain and swelling. The question was asked whether the muscle changes that occur due to larva migrans give rise to the autoimmune response. The presence of antistriational antibodies (aStrAbs) was determined in the following groups of patients: group 1--66 patients with toxocariasis and the presence of anti-Toxocara antibodies, group 2--22 patients suspected of Toxocara canis infection without anti-Toxocara antibodies, group 3--20 patients with active trichinellosis confirmed by anti-Trichinella antibodies. As control 25 healthy persons (group 4) were studied. The aStrAb were tested by the indirect fluorescence using unfixed cryostat sections of human striated muscle. The following results were obtained: group 1--42 sera positive, group 2--5 sera were weakly positive, group 3--all sera showed the presence of aStrAbs, group 4--no aStrAbs. The presence of aStrAbs in patients with parasitic infections may suggest the occurrence of anti-muscle autoimmune response.

The altered expression of MHC-class II determinants on monocytes of cancer patients.

SummaryThe expression of MHC class II determinants Ia.7 (detected by cross reactive mouse anti-Iak antibody) and HLA-DR on monocytes (MO) of gastric and colorectal cancer patients was examined. An increased proportion of MO bearing the Ia.7 determinant was found, while the number of MO expressing DR was not elevated. In gastric cancer patients the increased expression of the Ia.7 determinant was most pronounced in advanced cancer (stage IVA and IVB). The increased expression of this determinant was related to the presence of the tumour as the number of MO expressing Ia.7 decreased 6 months following surgical resection of the tumour. Further, the increased expression of Ia.7 on MO correlated with the tumour infiltration of the serosa. The Ia.7 determinants were mainly expressed on MO which also expressed the receptor for the Fc part of immunoglobulin. Immunostaining in cellular infiltrates surrounding the tumour revealed that Ia.7+ macrophages (MØ) were more numerous than in normal gastric mucosa and severe chronic gastritis and were mostly present in close proximity to tumour cells, while DR+ MØ were mainly localized within the stromal tissue of the tumour and their number was not increased in cancer infiltrates. These observations indicate that the Ia.7+ subpopulation of MØ may be involved in the anti-tumour response of the host.

Pharmacokinetics of a novel human intravenous immunoglobulin 10% in patients with primary immunodeficiency diseases: Analysis of a phase III, multicentre, prospective, open-label study

&NA; Intravenous immunoglobulin (IVIG) therapy is commonly used to treat patients with primary antibody deficiency. This prospective, open‐label, non‐randomised, multicentre, phase III trial investigated the pharmacokinetics of a new 10% liquid IVIG product (panzyga®; Octapharma) in 51 patients aged 2–75years with common variable immunodeficiency (n=43) or X‐linked agammaglobulinaemia (n=8). Patients were treated with IVIG 10% every 3 (n=21) or 4weeks (n=30) at a dose of 200–800mg/kg for 12months. Total immunoglobulin G (IgG) and subclass concentrations approximately doubled from pre‐ to 15min post‐infusion. The maximum concentration of total IgG (mean±SD) was 21.82±5.83g/L in patients treated 3‐weekly and 17.42±3.34g/L in patients treated 4‐weekly. Median trough IgG concentrations were nearly constant over the course of the study, remaining between 11.0 and 12.2g/L for patients on the 3‐week schedule and between 8.10 and 8.65g/L for patients on the 4‐week schedule. The median terminal half‐life of total IgG was 36.1 (range 18.5–65.9) days, with generally similar values for the IgG subclasses (26.7–38.0days). Median half‐lives for specific antibodies ranged between 21.3 and 51.2days for anti‐cytomegalovirus, anti‐Haemophilus influenzae, anti‐measles, anti‐tetanus toxoid, anti‐varicella zoster virus antibodies, and anti‐Streptococcus pneumoniae subtype antibodies. Overall, IVIG 10% demonstrated pharmacokinetic properties similar to those of other commercial IVIG 10% preparations and 3‐ or 4‐weekly administration achieved sufficient concentrations of IgG, IgG subclasses, and specific antibodies, exceeding the recommended level needed to effectively prevent serious bacterial infections. Graphical abstract: Symbol. No Caption available.

Thrombocytopenia in common variable immunodeficiency patients - clinical course, management, and effect of immunoglobulins.

Common variable immunodeficiency (CVID) is a primary immunodeficiency of humoral immunity with heterogeneous clinical features. Diagnosis of CVID is based on hypogammaglobulinaemia, low production of specific antibodies, and disorders of cellular immunity. The standard therapy includes replacement of specific antibodies with human immunoglobulin, prophylaxis, and symptomatic therapy of infections. High prevalence of autoimmunity is characteristic for CVID, most commonly: thrombocytopaenia and neutropaenia, celiac disease, and systemic autoimmune diseases. The study included seven children diagnosed with CVID and treated with immunoglobulin substitution from 2 to 12 years. Thrombocytopenia was diagnosed prior to CVID in four children, developed during immunoglobulin substitution in three children. In one boy with CVID and thrombocytopaenia, haemolytic anaemia occurred, so a diagnosis of Evans syndrome was established. Therapy of thrombocytopaenia previous to CVID included steroids and/or immunoglobulins in high dose, and azathioprine. In children with CVID on regular immunoglobulin substitution, episodes of acute thrombocytopaenia were associated with infections and were treated with high doses of immunoglobulins and steroids. In two patients only chronic thrombocytopaenia was noted. Splenectomy was necessary in one patient because of severe course of thrombocytopaenia. The results of the study indicated a supportive role of regular immunoglobulin substitution in patients with CVID and chronic thrombocytopaenia. However, regular substitution of immunoglobulins in CVID patients did not prevent the occurrence of autoimmune thrombocytopaenia episodes or exacerbations of chronic form. In episodes of acute thrombocytopaenia or exacerbations of chronic thrombocytopaenia, infusions of immunoglobulins in high dose are effective, despite previous regular substitution in the replacing dose.

The influence of fibronectin on proliferation and apoptosis of acute lymphoblastic leukaemia cells in vitro

The extracellular matrix (ECM) is a dynamic environment involved in the regulation of haematopoiesis. A crucial role of this structure is the promotion of proliferation, maturation, and differentiation of haematopoietic stem cells (HSC), and adhesion and migration of HSC in bone marrow. In the present study the effect of ECM proteins (fibronectin, collagens, laminin, thrombospondin, and vitronectin) on proliferation and apoptosis of acute lymphoblastic leukaemia cells isolated from acute lymphoblastic leukaemia (ALL) patients (in vitro) was assessed. The leukaemia cells were obtained as interphase on Ficoll/Isopaque (Pancoll human, PAN-Biotech) density gradient and, after washing, counted in a chamber. Subsequently, cells were used for culture and apoptosis assay. Presence of fibronectin, collagen type IV, and laminin was associated with inhibition of lymphoblastic leukaemia cell proliferation. Analysis of the culture of lymphoblastic leukaemia cells in the presence of ECM showed fibronectin as the most active protein.

Neurodegenerative changes detected by neuroimaging in a patient with contiguous X-chromosome deletion syndrome encompassing BTK and TIMM8A genes

Introduction In this study we describe a patient with gross deletion containing the BTK and TIMM8A genes. Mutations in these genes are responsible for X-linked agammaglobulinemia and Mohr-Tranebjaerg syndrome, respectively. X linked agammaglobulinemia is a rare primary immunodeficiency characterized by low levels of B lymphocytes and recurrent microbial infections, whereas, Mohr-Tranebjaerg syndrome is a progressive neurodegenerative disorder with early onset of sensorineural deafness. Material and methods For neuroimaging, the magnetic resonance imaging and magnetic resonance spectroscopy of the brain were performed. Microarray analysis was performed to establish the extent of deletion. Results The first clinical symptoms observed in our patient at the age of 6 months were connected with primary humoral immunodeficiency, whereas clinical signs of MTS emerged in the third year of live. Interestingly, the loss of speech ability was not accompanied by hearing failure. Neuroimaging of the brain suggested leukodystrophy. Molecular tests revealed contiguous X-chromosome deletion syndrome encompassing BTK (from exons 6 through 19) and TIMM8A genes. The loss of the patient’s DNA fragment was accurately localized from 100 601 727 to 100 617 576 bp on chromosome’s loci Xq22.1. Conclusions We diagnosed XLA-MTS in the first Polish patient on the basis of particular molecular methods. We detected neurodegenerative changes in MRI and MR spectroscopy in this patient. Our results provide further insight into this rare syndrome.