Biomedical subjects

K Poikonen

Publications and source records attributed to K Poikonen.

17 recordsLinked to original sources

PubMed2001

E2F mediates induction of the Sp1-controlled promoter of the human DNA polymerase epsilon B-subunit gene POLE2.

The B-subunits of replicative DNA polymerases from Archaea to humans belong to the same protein family, suggesting that they share a common fundamental function. We report here the gene structure for the B-subunit of human DNA polymerase epsilon (POLE2), whose expression and transcriptional regulation is typical for replication proteins with some unique features. The 75 bp core promoter region, located within exon 1, contains an Sp1 element that is a critical determinant of promoter activity as shown by the luciferase reporter, electrophoretic mobility shift and DNase I footprinting assays. Two overlapping E2F elements adjacent to the Sp1 element are essential for full promoter activity and serum response. Binding sites for E2F1 and NF-1 reside immediately downstream from the core promoter region. Our results suggest that human POLE2 is regulated by two E2F-pocket protein complexes, one associated with Sp1 and the other with NF-1. So far, only one replicative DNA polymerase B-subunit gene promoter, POLA2 encoding the B-subunit of DNA polymerase alpha, has been characterized. Mitogenic activation of the POLE2 promoter by an E2F-mediated mechanism resembles that of POLA2, but the regulation of basal promoter activity is different between these two genes.

Base Sequence↗

PubMed1999

SH3 domains with high affinity and engineered ligand specificities targeted to HIV-1 Nef.

The avid binding of HIV-1 Nef to the Src homology-3 (SH3) domain of Hck (KD 250 nM) has been shown to involve an interaction between the RT-loop of Hck-SH3 and residues in Nef outside of its prototypic polyproline type II (PPII) helix-containing SH3-ligand region. Such distinctive interactions are thought to provide specificity and affinity for other SH3/ligand protein complexes as well. Here, we have constructed and successfully displayed on the surface of M13 bacteriophage particles a complex library of SH3 domains, which are derived from Hck but carry a random hexapeptide substitution in their RT-loops (termed RRT-SH3). Using this strategy we have identified individual RRT-SH3 domains that can bind to Nef up to 40-fold more avidly than Hck-SH3. Some of these high-affinity RRT-SH3 domains resembled Hck-SH3 in that they bound much less well to a Nef variant containing an engineered F90R mutation that interferes with docking of the native Hck RT-loop. In addition, we could also select RRT-SH3 domains with an opposite specificity, which were dependent on the Arg90 residue for strong binding, and bound 100-fold less well to unmodified Nef. These results demonstrate the utility of phage-display in engineering of signaling protein interaction domains, and emphasize the importance of the RT-loop in SH3 ligand selection, thus suggesting a general strategy for creating SH3 domains with desired binding properties.

Amino Acid Sequence↗

PubMed1996

T-cell subpopulations in the cerebrospinal fluid and peripheral blood of patients with multiple sclerosis: a follow-up study.

We analyzed the relative and absolute numbers of CD4+ cells positive and negative for CD45RA marker as well as the numbers of CD8+ cells positive and negative for CD11b in the cerebrospinal fluid (CSF) and peripheral blood (PB) of 17 patients with multiple sclerosis (MS) during and 60 days after an exacerbation. We also studied samples from 27 control patients, 17 of whom had a noninflammatory neurologic disease and 10 of whom had an inflammatory neurologic disease other than MS. The results confirmed the small number of CD45RA-positive (naive or suppressor inducer) CD4+ cells as well as CD11b-positive (suppressor) CD8+ cells in the CSF compared with the PB for all paired CSF-blood comparisons. The relative numbers of these cells did not significantly differ between samples taken during an exacerbation and those taken 2 months later. In PB, the percentage of CD8+ CD11b-positive lymphocytes was lower in patients with MS or in patients with an inflammatory neurologic disease than in patients with a noninflammatory neurologic disease. The results indicate that there are fewer cells responsible for suppression-related phenomena in CSF than in PB but that this phenomenon is not specific to MS.

Adult↗

PubMed1995

Influx of nonactivated T lymphocytes into the cerebrospinal fluid during relapse of multiple sclerosis.

T lymphocytes were analyzed in the cerebrospinal fluid of 17 patients with multiple sclerosis during and 2 months after an exacerbation. The percentage of activated (HLA-DR+) T lymphocytes was decreased in the cerebrospinal fluid during the relapse but returned thereafter to the level comparable to that in patients with noninflammatory neurological diseases, whereas no fluctuation in the total number of these cells was found. This suggests an influx of nonactivated T lymphocytes into the cerebrospinal fluid at the beginning of the relapse.

Adult↗

PubMed1994

Application of fine-needle aspiration to the demonstration of ERBB2 and MYC expression by in situ hybridization in breast carcinoma.

Thirteen consecutive fine-needle aspirates of breast carcinoma and five selected breast tumor cell lines were analyzed for ERBB2 and MYC mRNA expression by in situ hybridization. To compare the level of mRNA synthesis with those of gene amplification and oncoprotein synthesis, all tumors were also analyzed by Southern blot analysis, and for ERBB2 also by immunohistochemistry. Expression of ERBB2 mRNA was observed in eight tumors. MYC expression was observed in all tumors studied. Three tumor cell lines expressed both ERBB2 and MYC (SK-BR-3, HeLa, HT-29) and two only MYC (SK-LU-1, HL-60). Only one tumor showed amplification of ERBB2 and two of MYC. In all three cases there was a considerable increase in corresponding mRNA synthesis as detected by in situ hybridization. By immunohistochemistry, four cases showed either patchy areas or uniformly distributed, membrane-bound ERBB2 immunoreactivity. All except one case showed increased ERBB2 mRNA synthesis. There was a clear association between the quantity of ERBB2 mRNA and oncoprotein expression. The results show that in situ hybridization of fine-needle aspiration material is a sensitive method to detect increased expression of the ERBB2 and MYC oncogenes in breast carcinoma. Furthermore, this study indicates that in a majority of cases some other mechanism that gene amplification appears responsible for the increased gene expression. It is also possible that Southern blot analysis is not a sensitive enough method to detect gene amplifications in the heterogeneous breast tumors, which usually also contain stromal tissue. The fact that not all cases with elevated ERBB2 mRNA synthesis were immunohistochemically positive suggests that either immunohistochemistry (after fixation with 10% formalin) is a less sensitive method than in situ hybridization to detect abnormal gene expression or that there are cases in which the oncoprotein synthesis is for some reason depressed, even though there is an increase in gene transcription.

Adult↗

PubMed1991

Characterization of nickel-specific T cell clones.

Nickel is the major cause of metal-induced allergic dermatitis. Twelve nickel-specific T cell clones were used to investigate the cellular immune reactions occurring in nickel sensitivity. The selection between the alternative T cell receptors alpha beta and gamma delta and two alternative V beta genes (V beta 5 and V beta 8) were studied to see if nickel induces a selective pressure for clones bearing particular genes. Cell surface markers were studied by monoclonal antibodies and flow cytometry. Soluble mediators were measured by an ELISA method. The clones used T cell receptor alpha beta genes but did not use V beta 5 or V beta 8. They were T helper clones with a primed memory marker (CD3+ CD4+ CD8- CD45RO+) and carried HLA-DR. None of the clones secreted IL-1 alpha, all of them secreted IL-2 receptor. Four clones secreted IL-1 beta, six IL-4 and seven IL-6, the peaks in IL-2R and IL-6 secretion preceding IL-4 secretion. The clones helped immunoglobulin synthesis. The clones from late effector phase of the nickel allergic reaction favours the use of T cell receptors alpha beta genes. Nickel-specific clones were phenotypically indistinguishable but differed in soluble mediators produced.

Antigens, CD↗

PubMed1990

Helicobacter pylori induces lymphocyte activation in peripheral blood cultures.

Helicobacter pylori-induced in vitro stimulation of mononuclear cells was characterized by measuring DNA synthesis response, interferon-gamma (IFN-gamma) secretion and the number of immunoglobulin-secreting cells. The strength of these responses was measured in 51 subjects comprising 36 dyspeptic patients from the Gastroenterological Unit and 15 members of the laboratory staff. Nineteen subjects had antibodies to H. pylori and 32 did not. The responses were compared with respect to H. pylori antibody status. Positive stimulation of DNA synthesis (stimulation index greater than 2) was obtained in 96% of the subjects, and the bacterium induced IFN-gamma secretion in all of them. The induction of immunoglobulin-secreting cells revealed B cell in vitro stimulation. The antibody-positive subjects had a tendency to synthesize less DNA (P not significant) and secrete less IFN-gamma (P less than 0.05) in response to H. pylori than did the antibody-negative ones, but the differences were not marked. The results show that the whole H. pylori bacterium stimulates mononuclear cells. The nature of the stimulation is not yet characterized (non-specific versus specific).

Antibodies, Bacterial↗

PubMed1990

Analysis of impaired in vitro immunoglobulin synthesis in rheumatoid arthritis.

Decreased immunoglobulin production in pokeweed mitogen driven lymphocyte cultures has been reported in rheumatoid arthritis (RA). Here various activators and experimental designs have been used to determine the contribution of B cells, T cells, or monocytes to this low response. Sixty patients with RA and paired controls were studied at the onset of disease and again six months later. Concentrations of IgA, IgG, and IgM in cultures of RA peripheral blood mononuclear cells stimulated with thymus dependent activators were already decreased at the onset of the disease. Six months later RA mononuclear cells produced even lower concentrations of immunoglobulin. In contrast, stimulation with a T cell independent activator showed that RA B lymphocytes had retained normal potential to synthesise immunoglobulin. Poor helper function was indicated by costimulation experiments and cultures of mixed mononuclear cells from patients and controls. This notion was supported also by the fact that phytohaemagglutinin induced interleukin-2 production by RA mononuclear cells was less than half of the control values. Nonspecific suppressor activity was similar in RA and controls. Monocyte functions were normal when tested by addition of indomethacin or 2-mercaptoethanol to the mitogen activated cultures. The defect in mitogen stimulated immunoglobulin production in vitro of RA mononuclear cells thus was more pronounced with time and probably reflects impaired mediator associated help in the differentiation of B lymphocytes into immunoglobulin secreting cells.

Adolescent↗

PubMed1989

Salivary immunoglobulins, lysozyme, pH, and microbial counts in children receiving anti-neoplastic therapy.

Salivary immunoglobulins, lysozyme, pH, and microbial counts were determined in 55 children with cancer diseases (37 cured subjects and 18 acute ones) and 103 healthy subjects. 5-10 ml unstimulated whole saliva was collected and pH, immunoglobulins and lysozyme were measured. Chairside dip-slide cultivations were used for microbiologic cultures. Reduced salivary pH and an increased amount of lysozyme were found in the saliva of those children who had been cured of their cancer diseases, but ongoing cancer disease or the treatment provided for it reduced pH and increased the amounts of lysozyme, lactobacillus, Streptococcus mutans and some immunoglobulins. These findings suggest that children with childhood cancer may be more susceptible to dental diseases than healthy ones.

Adolescent↗

PubMed1989

Francisella tularensis-specific T-cell clones are human leukocyte antigen class II restricted, secrete interleukin-2 and gamma interferon, and induce immunoglobulin production.

T-cell clones (TLC) were established from a Francisella tularensis-vaccinated subject in order to study the cells responsive for cell-mediated immunity against F. tularensis. All the clones were human leukocyte antigen (HLA) class II restricted to one of the HLA-DR specificities of the original donor. The TLC cells were CD4+ and produced interleukin-2 and gamma interferon after stimulation with specific antigen. Seven of the eight clones tested assisted in the production of immunoglobulin G (IgG), IgA, and IgM antibodies.

Clone Cells↗

PubMed1987

Characterization of in vivo activated lymphocytes found in the peripheral blood of patients undergoing cardiac operation.

We studied in vivo lymphocyte activation in patients who underwent cardiac operation. A large number of deoxyribonucleic acid-synthesizing cells characterized by 3H-thymidine uptake and morphologically atypical lymphocytes were found after operation in the peripheral blood samples of patients, the peak level occurring on the sixth or seventh postoperative day. Most of the deoxyribonucleic acid-synthesizing cells were in the B cell fraction. The number of antibody secreting cells in the patients' peripheral blood samples was found increased from the normal level after operation. Immunoglobulin A-secreting cells were the most elevated fraction. The percentage of helper (OKT4) and suppressor (OKT8) cells in the postoperative samples was not significantly different from that in the preoperative samples. Only a few were Ia antigen positive cells. Co-culture of patients' B cells with their own T cells and with the T cells taken from two healthy controls revealed no signs of any helper or suppressor effect on these antibody-secreting cells. The results show that after cardiac operation the peripheral blood of patients contains an increased number of immunoglobulin secreting cells. The regulatory T-lymphocytes seem not to have any effect in vitro on these in vivo activated cells.

Adult↗

PubMed1986

Nocardia water soluble mitogen (NWSM), a human B-lymphocyte mitogen.

Nocardia water soluble mitogen (NWSM), which is a peptidoglycan, was prepared from Nocardia opaca. Its effect on human B-lymphocyte proliferation, the number of immunoglobulin secreting B-cells, and bone marrow stem cell growth was studied in vitro. NWSM stimulated the proliferation of B-lymphocytes of all ten healthy subjects studied very effectively (P less than 0.0005). No T-cell activation occurred. NWSM did not increase the amount of antibody producing cells. The growth of bone marrow stem cells was enhanced only in one patient out of the eight studied. Thus NWSM is a B-lymphocyte mitogen with a potential effect on hematopoietic stem cells. However, production of NWSM was faced with difficulties because not all lots prepared had B-cell stimulating activity.

Antibody-Producing Cells↗

PubMed1986

Effect of elevated temperature on human immunoglobulin synthesis, lymphokine production and lymphocyte proliferation in vitro.

Fever in infections is mediated by endogenous pyrogen, a protein inseparable from interleukin 1 (IL-1). It remains unclear what the role of fever in host defence is. We studied the effect of elevated temperature on mitogen-induced human lymphocyte stimulation in vitro. Lymphocyte proliferation, elaboration of leukocyte migration inhibiting factor (LIF) and immunoglobulin production were measured at normal and elevated temperatures. Pokeweed mitogen-(PWM) driven responses were quantitated with a plaque-forming cell assay and by measuring secreted immunoglobulins in culture medium. The responses at 38, 39 or 40 degrees C were, if anything, lower than those at 37 degrees C. The production of LIF after stimulation with PWM was not augmented at elevated temperatures. In contrast, the proliferative responses to PWM, phytohaemagglutinin (PHA) and concanavalin A (ConA) were increased at 38 degrees C. These results indicated that elevated temperature has diverse effects on mitogen-induced lymphocyte functions in vitro.

Cells, Cultured↗

PubMed1986

Experimental intestinal infection of rats by Yersinia enterocolitica 0:3. A follow-up study with specific antibodies to the virulence plasmid specified antigens.

Rats were infected by intragastric inoculation of Yersinia enterocolitica 0:3 grown at room temperature. The events of the first 6 days of the infection were followed by staining sections of small intestine using an immunoperoxidase method. The specific antibodies used were either rabbit antibodies to antigens of Y. enterocolitica, to the temperature-inducible antigens specified by the virulence plasmid (pYV) of Y. enterocolitica, or monoclonal antibody to the pYV-specified autoagglutination protein P1. In the course of the infection Y. enterocolitica organisms were detectable in the terminal ileum already 1 h after the challenge. By 1 h the pYV-specified temperature-inducible antigens were expressed in Y. enterocolitica both in the lumen of the intestine and in the intestinal tissues, which indicates that the activation of pYV in vivo was very rapid. Y. enterocolitica organisms which had penetrated into the lamina propria of the villi were rapidly removed by phagocytic cells. Later organisms were located intracellularly in the lamina propria, in Peyer's patches and in the regional lymph nodes. The terminal ileum was the most severely affected part of the small intestine.

Animals↗

PubMed1985

Synthesis of human immunoglobulins in vitro: comparison of two assays of secreted immunoglobulin.

One consequence of B-lymphocyte activation is immunoglobulin production, which can be quantitated by various techniques. We have compared assay of plaque forming cell (PFC) and determination of immunoglobulins by ELISA in culture supernatants of human lymphocytes stimulated with pokeweed mitogen and with Staphylococcus aureus. These assays correlated well (r greater than 0.77) in all major immunoglobulin classes studied. The close correlation suggests that determination of secreted immunoglobulins by ELISA may be substituted for the PFC assay.

B-Lymphocytes↗

PubMed1983

Selenium and immune functions in humans.

Earlier animal experiments have shown that selenium depletion may decrease immune functions. In this human study, 40 volunteers from a population with low serum selenium concentrations were supplemented with selenium or placebo for 11 weeks. Blood samples were drawn at intervals for analysis of selenium status and immune function. At the end of the supplementation period, plasma selenium levels were 74 ng/ml in the placebo group and 169 ng/ml in the supplemented group. The improvement in selenium status was associated with a 57% increase in the activity of platelet glutathione peroxidase in the group supplemented with selenium, but there was no increase in the activity of this enzyme in the placebo-treated subjects. Immune function was measured in vitro by tests of lymphocyte and granulocyte activity. Intracellular killing of Staphylococcus aureus by granulocytes was slightly lower in the placebo group than in the selenium group at the end of the supplementation period (77.2 compared to 85.2%; P less than 0.05). No significant changes were observed in phagocytosis, chemotactic factor generation, antibody or leukocyte migration inhibitory factor production by lymphocytes, or proliferative responses to phytohemagglutinin or concanavalin A. These results suggest that the selenium deficiency of the order found in Finland and some other areas of the world has little, if any, influence on the immune functions measured in this study.

Adult↗

PubMed1982

Synthesis of IgM, IgG and IgA in rheumatoid arthritis.

We studied the production of immunoglobulins by lymphocytes separated from the blood of 15 rheumatoid arthritis (RA) patients, of 12 patients suffering from other connective tissue diseases (CTD), and of 18 healthy controls. The production of IgM, IgG and IgA in pokeweed-mitogen-stimulated cultures was measured by counting the number of plaque-forming cells (PFC) and by determining the concentration of secreted immunoglobulins by means of an enzyme immunoassay. Synthesis of immunoglobulins, particularly IgM and IgG, was lower than in other CTD patients or controls. The IgM response of RA patients was 20% and 29% (PFC and Ig concentrations) that of the controls. The respective figures for IgG were 33% and 53% and for IgA 61% and 72%.

Adult↗