Time and Temperature Stability of TGF-ß1, EGF and IGF-1 in 20% and 100% Human Serum.

Autologous serum eye drops (ASEDs) are used as a treatment for severe dry eye disease. The concentration and stability of various growth factors in ASEDs is determinative for their efficiency. We therefore assessed the concentrations of transforming growth factor beta 1 (TGF-ß1), epidermal growth factor (EGF) and insulin-like growth factor 1 (IGF-1) in ASEDs following storage at 4-8, -20, -80 and -156 °C. Twenty % and 100% sera from eight healthy volunteers were analysed by the sandwich enzyme immunoassay at different time intervals up to seven months. The mean levels of TGF-ß1 and EGF in undiluted and 20% serum did not differ significantly from the baseline levels in fresh serum for any storage conditions after 7 days at 4-8 °C, as well as after 4- and 7-month preservation at sub-zero temperatures. In 20% serum, no IGF-1 concentration decrease was found following 7 days of preservation at 4-8 °C. However, a decrease to 78 % and 81 % (P < 0.01) of baseline values was found in 20% serum after 4-month storage at -20 °C and 7-month storage at -156 °C, respectively. A more pronounced decrease in IGF-1 was observed in undiluted serum. All assessed growth factors present in 20% frozen serum remained stable for up to 7 months. The highest stability was achieved at -80 °C. At -20 and -156 °C, some decrease in IGF-1 occurred. Our results indicate that 20% ASEDs can be stored frozen up to 7 months under proper conditions.

[Tear Osmolarity in Patients with Severe Dry Eye Syndrome Before and After Autologous Serum Treatment: a Comparison with Tear Osmolarity in Healthy Volunteers]. / Osmolarita slz u pacientu s tezkým syndromem suchého oka pred a po aplikaci autologního séra. Porovnání s hodnotami zdravých dobrovolníku.

INTRODUCTION: Dry eye syndrome (DES) is a multifactorial disease of the tears and ocular surface. Recently, treatment with autologous serum eye drops (AS-ed) has been frequently used in these patients. Significant improvement correlates well with clinical, laboratory and subjective findings. It is assumed that one of the key factors in the development of the disease is increased tear osmolarity. The aims of our study were to test tear osmolarity measurements in clinical practice, to examine if osmolarity values differ before and after a 3-month application of 20% AS-ed, and to determine if the values differ between patients with severe DES and healthy individuals. METHODS: The study included 35 patients with severe DES (Schirmer test<5 mm/5 min) and 23 healthy volunteers. Tear osmolarity values (TearLab Osmolarity System), the Schirmer test (ST1), vital ocular surface staining and subjective feelings (the OSDI questionnaire) were assessed in patients with DES before and after treatment with 20% AS-ed. Further, the tear osmolarity values were compared between healthy subjects and patients with DES before and after treatment with AS-ed. RESULTS: The values of OSDI and vital staining significantly decreased in patients with DES after the treatment (p<0.0001). ST1 and tear osmolarity did not change significantly after the treatment. ST1 values in healthy individuals were significantly higher (p<0.0001) and the OSDI values significantly lower (p<0.0001) than the results obtained in patients before and after treatment. Tear osmolarity was not statistically different between healthy subjects (306 mosmol/l) and patients with DES (302 and 301 mosmol/l before and after treatment respectively). CONCLUSION: We demonstrated a positive effect of AS treatment on the ocular surface in patients with DES. However, the osmolarity values did not differ before and after treatment with AS, and they also did not differ significantly between DES patients and healthy individuals. In accordance with other recent studies, our results raise questions concerning the value of the TearLab Osmolarity System for evaluating therapeutic effect and also as a tool for DES diagnosis.

Failure of multiple antivirals to affect high HHV-6 DNAaemia resulting from viral chromosomal integration in case of severe aplastic anaemia.

We report a fifty-year-old woman presenting with severe aplastic anaemia (SAA) and prolonged high Human Herpesvirus 6 (HHV6) variant A DNAeamia detected by quantitative PCR. Multiple antiviral treatments failed to affect the HHV6 DNAemia and subsequent immunosuppressive treatment reached only partial improvement as judged by bone marrow examinations. The patient remained dependent on thrombocyte transfusions and G-CSF treatment. After one year of steady high HHV6 DNA load in blood, viral chromosomal integration was proved by demonstrating the viral DNA in hair follicles. This condition appeared to be unconnected with, and to have no effect, on the original SAA.

[Gene therapy of the graft versus host reaction]. / Genová terapie reakce stepu proti hostiteli.

Graft-versus-host-disease (GVHD) is a frequent and dangerous complication of allogenic transplantations of bone marrow. Gene therapy offers a way to deal with the problem. It is based on the introduction of suicide genes (SG) into the donor's T lymphocytes, which are responsible for the development of GVHD. If it develops, the presence of SG in the effector cells gives an opportunity to get rid of them, because their products are capable of changing otherwise innocuous substances into highly cytotoxic metabolites. For the transduction of SG retrovirus-based vectors are used. The authors tried to employ for this purpose recombinant adeno-associated viruses (rAAV). The attempt was unsuccessful. When using rAAV as vectors, the efficacy of transduction was very low. Further experiments indicated that this failure was due to the absence of receptor for AAV in T lymphocytes. It seems clear that until the surface of rAAV is modified to facilitate their penetration into T cells, they cannot replace retroviruses for transfer of SG into this cell type.

Comparison of two different methods for CD34+ selection and T cell depletion in peripheral blood stem cell grafts--our experiences with CellPro, E rosetting and CliniMACS technique.

The aim of this study was to establish a suitable method for in vitro T cell depletion in peripheral blood stem cell grafts for mismatched/haploidentical transplantation in children and adults with severe hematological disorders and for autologous transplantation in patients with autoimmune diseases refractory to conventional immunosuppressive treatment. Two different selection techniques have been used: CD34+ selection using immunoaffinity columns (CellPro Ceprate) followed by T cell depletion by E-rosetting or CD34+ selection using submicroscopic paramagnetic beads (CliniMACS device) with T cell depletion in a one step procedure. The mean purity and recovery of CD34+ cells and efficiency of T cell removal in the final product were compared. From March 1995 to December 1998 we prepared twelve allografts using Cell Pro system for eight children with high-risk hematological malignancies and six autografts for six patients with severe autoimmune diseases. From January 1999 to October 2000 we prepared fifteen allografts using CliniMACS system for ten children with high-risk hematological diseases and inborn metabolic disorders or primary immunodeficiences, five allografts for three adult patients with high-risk hematological malignancies and two autografts for two patients with autoimmune diseases. In allogeneic transplantation the median purity of CD34+ cells in the final products after CellPro and E-rosetting was 85.6% (55.3%-95.7%); median recovery was 24.8% (17%-35%), median transplanted doses of T cells per kilogram of body weight were 0.66x10(4) (0-2.8); in autologous transplantation the median purity of CD34+ was 92.6% (55.6%-96%), median recovery was 28% (22%-46.2%), median transplanted doses of T cells per kilogram of body weight were 0.39x10(4) (0.0-3.6). After CliniMACS technique the median purity of CD34+ cells was 94.87% (69.15%-99%),medianrecoverywas 58% (30%-79.6%), median transplanted doses of T cells per kg of body weight were 0.254x10(4) (0-14.15); in autologous transplantation the median purity of CD34+ was 94% (94%-94%, median recovery was 97.4% (95%-99.8%), median transplanted doses of T cells per kilogram of body weight were 0.87x10(4) (0.49-1.24). We consider both methods of CD34+ selection and T cell depletion suitable for peripheral blood stem cell processing before mismatched hemopoietic stem cell transplantation in patients without identical donor or before autologous transplantation for severe autoimmune diseases. However, magnetic separation using CliniMACS system results in higher levels of purity and recovery with efficient T cell depletion.

[Purging of hemopoietic progenitor cells in autologous transplantation]. / Cistení hemopoetických progenitorových bunek pro autologní transplantace.

The authors describe the results of purification of bone marrow and peripheral progenitor cells (PBPC) for clinical transplantations. Vepeside was used to purify in 1990-1995 a total of 41 bone marrows of adults and children. Of these 23 were transplanted. Maphosphamide was used bone marrow purging in two patients; transplantation was performed in one case. By a combination of Vepeside with methylprednisolone haematopoietic cells of 24 patients were purged, transplantations were performed in 10. Three-day cultivation of haematopoietic cells in the presence of Desferal was used for purging cells of 22 patients with neuroblastoma; transplantations were performed in 10 patients. The authors give the values of nucleated cells, haematopoietic colonies of CFU-GM and CD34 positive cells obtained after purification calculated per kg body weight of the patient and the percentage yields.