Initial experience with lung transplantation in Estonia.

BACKGROUND: A lung transplant program in Estonia was initiated in cooperation with the Medical University of Vienna. The first lung transplantation for an Estonian patient was performed in Vienna on April 28, 2009. The waiting list in Estonia was opened on May 28, 2010; the first transplantation was performed on October 7, 2010. The aim of this study was to present our initial results. PATIENTS AND METHODS: All lung transplantations performed in Estonia through the end of January 2012 included 2 female and 3 male patients of age from 52 to 64 years. Data regarding the donor, the transplant operation, postoperative period, and follow-up were extracted from case records. RESULTS: The cases included 1 bilateral lobar, 3 double, and 1 single lung transplantations. Two patients had chronic obstructive pulmonary disease, one alpha-1 trypsin deficiency, and two idiopathic pulmonary fibrosis. The operative duration varied from 172 to 337 minutes; the ischemia times for the first and second lung ranged from 191 to 351 and 303 to 455 minutes, respectively. Duration of postoperative mechanical ventilation ranged from 2 to 14 days (median 3) and the hospital stay from 28 to 72 days. The following complications were observed: prolonged air leak in 2 patients, one of whom required rethoracotomy; phrenic nerve palsy in 2, atrial fibrillation in 2, and mild renal failure in 1 subject. One patient needed readmission to the intensive care unit owing to acute respiratory failure; one, a tracheostomy for weaning from the ventilator, and one, noninvasive ventilation owing to hypercapnia. All patients remain well at 4-19 months after transplantation. No episodes of acute rejection or bronchiolitis obliterans have been diagnosed. CONCLUSION: The first 1.5-year experience with lung transplantation in Estonia has been satisfactory. Although there have been several complications, no posttransplant or waiting list mortality has occurred.

Intercomparison of Rn-222 determination from groundwater.

An intercomparison exercise on Rn-222 determination in groundwater was organized between eight Nordic laboratories. The individual laboratory results were in most cases within 20% of the median value and within reported uncertainties. Considering the particular difficulties in preparing, transporting and analyzing Rn-222, being a gaseous radionuclide, the results indicate a high analytical capability among the Nordic laboratories. In order to maintain a high analytical quality, similar intercomparisons will also be needed in the future.

Cytokine production in nickel-sensitized individuals analysed with enzyme-linked immunospot assay: possible implication for diagnosis.

BACKGROUND: Patients with suspected allergic contact dermatitis still have to undergo patch testing for a correct diagnosis. As this has several disadvantages there is a need for additional methods, preferentially those that can be performed in vitro. Objectives To investigate the possibility of diagnosing contact allergy to nickel (Ni2+) using the enzyme-linked immunospot (ELISpot) assay that allows the analysis of cytokines at a single-cell level in ex vivo activated peripheral blood mononuclear cells (PBMC). METHODS: Eleven female patients and nine age- and sex-matched healthy volunteers participated in the study. All patients had a history of nickel allergy and a positive patch test reaction to NiSO4, while the controls' test was negative. PBMC were cultured in the presence or absence of NiCl2. Cell proliferation was measured with [3H]thymidine incorporation, and the number of cytokine-producing cells analysed with the ELISpot assay. RESULTS: The proliferative response of PBMC to Ni2+, expressed as stimulation index, was significantly higher in the nickel-allergic patients than in the control group. Using the ELISpot assay, we found that PBMC from nickel-allergic individuals responded to Ni2+ with significantly greater production of interleukin (IL)-4, IL-5, IL-13 and interferon-gamma, but not IL-12, compared with the healthy controls. The number of IL-4- and IL-5-producing cells correlated with the number of IL-13-producing cells in the nickel-allergic patients, but Ni2+-induced PBMC proliferation did not correlate with the number of cytokine-producing cells for any of the cytokines tested. CONCLUSIONS: Our results indicate that the ELISpot assay could be a tool in the discrimination between nickel-allergic and non-allergic individuals.

Antigen loading of MHC class I molecules in the endocytic tract.

Major histocompatibility complex (MHC) class I molecules bind antigenic peptides that are translocated from the cytosol into the endoplasmic reticulum by the transporter associated with antigen processing. MHC class I loading independent of this transporter also exists and involves peptides derived from exogenously acquired antigens. Thus far, a detailed characterization of the intracellular compartments involved in this pathway is lacking. In the present study, we have used the model system in which peptides derived from measles virus protein F are presented to cytotoxic T cells by B-lymphoblastoid cells that lack the peptide transporter. Inhibition of T cell activation by the lysosomotropic drug ammoniumchloride indicated that endocytic compartments were involved in the class I presentation of this antigen. Using immunoelectron microscopy, we demonstrate that class I molecules and virus protein F co-localized in multivesicular endosomes and lysosomes. Surprisingly, these compartments expressed high levels of class II molecules, and further characterization identified them as MHC class II compartments. In addition, we show that class I molecules co-localized with class II molecules on purified exosomes, the internal vesicles of multivesicular endosomes that are secreted upon fusion of these endosomes with the plasma membrane. Finally, dendritic cells, crucial for the induction of primary immune responses, also displayed class I in endosomes and on exosomes.

Follicular dendritic cells carry MHC class II-expressing microvesicles at their surface.

Follicular dendritic cells (FDCs) present in lymphoid follicles play a critical role in germinal center reactions. They trap native Ags in the form of immune complexes providing a source for continuous stimulation of specific B lymphocytes. FDCs have been reported to express MHC class II molecules, suggesting an additional role in the presentation of not only native, but also processed Ag in the form of peptide-loaded MHC class II. Adoptive bone marrow transfer experiments have shown that MHC class II molecules are only passively acquired. Up to now the origin of these MHC class II molecules was not clear. Here we show by cryoimmunogold electron microscopy that MHC class II molecules are not present at the plasma membrane of FDCs. In contrast, microvesicles attached to the FDC surface contain MHC class II and other surface proteins not expressed by FDCs themselves. The size and marker profiles of these microvesicles resemble exosomes. Exosomes, which are secreted internal vesicles from multivesicular endosomes, have been shown earlier to stimulate proliferation of specific T lymphocytes in vitro, but their target in vivo remained a matter of speculation. We demonstrate here that isolated exosomes in vitro bind specifically to FDCs and not to other cell types, suggesting that FDCs might be a physiological target for exosomes.

The radiological exposure of man from radioactivity in the Baltic Sea.

A radiological assessment has been carried out considering discharges of radioactivity to the Baltic Sea marine environment since 1950. The sources of radioactivity that have been evaluated are atmospheric nuclear-weapons fallout, fallout from the Chernobyl accident in 1986, discharges of radionuclides from Sellafield and La Hague transported into the Baltic Sea, and discharges of radionuclides from nuclear installations located in the Baltic Sea area. Dose rates from man-made radioactivity to individual members of the public (critical groups) have been calculated based on annual intake of seafood and beach occupancy time. The dose rates to individuals from the regions of the Bothnian Sea and Gulf of Finland are predicted to be larger than from any other area in the Baltic Sea due to the pattern of Chernobyl fallout. The dose rates are predicted to have peaked in 1986 at a value of 0.2 mSv year-1. Collective committed doses to members of the public have been calculated based on fishery statistics and predicted concentrations of radionuclides in biota and coastal sediments. The total collective dose from man-made radioactivity in the Baltic Sea is estimated at 2600 manSv, of which approximately two-thirds originate from Chernobyl fallout, approximately one-quarter from atmospheric nuclear-weapons fallout, approximately 8% from European reprocessing facilities, and approximately 0.04% from nuclear installations bordering the Baltic Sea area. An assessment of small-scale dumping of low-level radioactive waste in the Baltic Sea in the 1960s by Sweden and the Soviet Union has showed that doses to man from these activities are negligible. Dose rates and doses from natural radioactivity dominate except for the year 1986 where dose rates to individuals from Chernobyl fallout in some regions of the Baltic Sea approached those from natural radioactivity.

Stimulation of CD40 in human bladder carcinoma cells inhibits anti-Fas/APO-1 (CD95)-induced apoptosis.

CD40 and the CD95 (Fas/APO-1 antigen) are both members of the tumor necrosis factor receptor family. Whereas CD40 mediates a strong growth stimulatory signal in B cells, engagement of the CD95 receptor leads to growth inhibition and induction of apoptosis. As it has been reported that CD40 activation may rescue B cells from undergoing apoptosis, we were interested to see whether it had a similar effect in other cells expressing the CD40 receptor. We used epithelial tumor cells from the urinary bladder, a cell type that frequently expresses CD40 but for which no clear function of the molecule has been assigned. We found that the ligation of CD95 with the antibody anti-APO-1 induced apoptosis in most of the cell lines tested. Stimulation of CD40 with antibodies or a soluble construct of the CD40 ligand was shown to protect cells from apoptosis, as demonstrated by their ability to suppress the growth inhibition exerted by the anti-APO-1 antibody. Our results show that CD40 stimulation make cells less vulnerable to apoptosis induced via CD95 and suggest that CD40 expression on epithelial tumors may be associated with cell survival.

Adaptation of human bladder carcinoma cell lines to serum-free growth. Evidence for autocrine growth stimulation.

Tumor transformation is associated with a partial breakdown of the normal regulatory systems governing cell proliferation and differentiation. As a consequence, malignant cells are often less dependent on external growth factors and may be refractory to differentiation signals. Consistent with this view, we here show that 5 of 9 human bladder carcinoma cell lines (5637, HU549, SD, TCCSuP and T24) as well as a colon carcinoma line, HCT-8, and the melanoma line, HS294T, could be adapted to grow continuously in medium without serum or any other source of protein. The cells grown under these conditions displayed a longer generation time and were more dependent on a high initial cell concentration for survival and outgrowth. However, in most other respects, including cell morphology, growth pattern and antigenic phenotype, the cells were very similar to the original cultures. Conditioned medium from all the cell lines of bladder tumor origin as well as the HCT-8 colon carcinoma line was shown to contain autocrine growth stimulatory activity. Furthermore, criss-cross experiments showed that supernatants stimulated not only proliferation of the autologous cell line but also growth of the other cell lines, suggesting the production of a common autocrine factor/s in bladder tumor cells. Incubation of cells with radioiodinated supernatant allowed the identification of several candidate molecules for this factor activity. The study supports previous observations of autocrine stimulation as a mechanism for tumor cells to acquire autonomous growth capacity and indicates that this may be an important element in the oncogenesis of bladder tumors.

Agonistic properties of anti-B cell antibodies purified on staphylococcal protein A may be due to contaminating protein A.

Some antibodies directed to cell surface receptors may mimic the physiological ligands by inducing the transmission of activation or growth signals. Such agonistic antibodies have proven very useful when studying functional properties of various receptor molecules on, e.g., lymphoid cells. However, while investigating the agonistic effects on tonsillar B cells of the anti-CD43 monoclonal antibody (mAb) D4B11 and the anti-CD40 mAb S2C6, we made some observations which emphasize the need for caution when using antibodies purified by protein A affinity chromatography. Both antibody preparations were found to elicit changes in the intracellular free calcium concentration ([Ca2+]i) as well as promoting proliferation of phorbol ester activated cells. However, a closer analysis showed that the increase in [Ca2+]i could be attributed to soluble staphylococcal protein A (SpA) desorbed during antibody purification. By using pure soluble SpA, we were able to show that nanogram amounts were sufficient to increase [Ca2+]i by a mechanism that involved both a mobilization from intracellular stores and an influx across the B cell membrane. A similar effect on cytosolic Ca2+ in B cells was also noted for streptococcal protein G (protein G), another bacterial component used for antibody purification. However, in contrast to SpA, protein G had little effect on cell proliferation. These observations suggest that the presence of trace amounts of SpA or protein G in antibodies purified on these bacterial components may lead to incorrect interpretations of the agonistic properties of such antibodies. When the above findings were taken into account, it was found that the CD43 mAb D4B11, like the CD40 mAb S2C6, stimulated growth of B cells without causing any measurable increase in [Ca2+]i. Both CD40 and CD43 may thus be coupled to signalling pathways which do not involve breakdown of membrane phosphoinositides.

The human B lymphocyte and carcinoma antigen, CDw40, is a phosphoprotein involved in growth signal transduction.

The human B lymphocyte and carcinoma-associated Ag, CDw40, (p50, Bp50) is a receptor candidate for normal growth regulation. Interaction of mAb with this pan-B Ag, together with preactivating agents such as 12-O-tetradecanoylphorbol-13-acetate or anti-mu, deliver strong growth-promoting signals to the cells. We here demonstrate that signaling through this Ag is dependent on its aggregation on the cell surface. Thus, monovalent antibody fragments were relatively inefficient in this respect but effectively blocked stimulation by intact antibody. By using affinity purified CDw40 protein we have also demonstrated that it is antigenically distinct from other B cell-associated Ag, including the six differentiation clusters CD19 to CD24. The mAb S2C6 and G28.5, prepared by immunizing mice with human bladder carcinoma cells or tonsillar B-cells, respectively, were the only antibodies giving detectable binding. Either of these antibodies could also completely block the binding of the other, suggesting an identity or structural proximity of the epitopes recognized. The CDw40 Ag was shown to be a phosphoprotein lacking intrinsic protein kinase activity. The results provide further evidence for CDw40 being an important B cell growth factor receptor which may also have growth regulatory functions in the development of certain human carcinomas.