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Biomedical subjects

E Saksela

Publications and source records attributed to E Saksela.

At least 55 records · Page 3Linked to original sources
PubMed1991

Tumor necrosis factor as a potent inhibitor of adrenocorticotropin-induced cortisol production and steroidogenic P450 enzyme gene expression in cultured human fetal adrenal cells.

We have previously demonstrated that tumor necrosis factor alpha (TNF-alpha), a multifunctional cytokine mainly produced by activated monocytes, inhibits the ACTH-induced production of cortisol in cultures of human fetal adrenals. To clarify the molecular basis of this suppression, we investigated the effect of recombinant TNF-alpha (rTNF-alpha) on the messenger RNAs (mRNAs) for adrenal cytochrome P450 oxidases, P450scc (cholesterol side-chain cleavage enzyme/20.22-desmolase), P450c11 (11 beta-hydroxylase/18-hydroxylase/18-methyl oxidase), P450c17 (17 alpha-hydroxylase/17,20-lyase), and P450c21 (21-hydroxylase). Northern and dot blot experiments showed that 36 h incubation of primary cultures of human fetal adrenals with ACTH (200 ng/ml) increased the levels of all P450 enzymes severalfold. Preincubation of the cultures with rTNF-alpha at concentrations ranging from 0.1-100 ng/ml produced a dose-dependent inhibition of the ACTH-induced accumulation of all P450 mRNAs. The decrease in the expression of genes for steroidogenic enzymes was accompanied by a similar decrease in the production of cortisol but not in that of dehydroepiandrosterone sulphate nor androstenedione. Neither the basal expression of P450 enzymes nor the basal secretion of the steroids was significantly altered by 10 ng/ml of rTNF-alpha. rTNF-alpha did not affect the level of actin mRNA, the cell viability, nor the cell number. All the effects brought about by rTNF-alpha could be neutralized by addition of monoclonal anti-TNF-alpha antibody. These results show that TNF-alpha suppresses the synthesis of cortisol and shifts the steroid secretory pattern towards androgen production at least partly by suppressing the accumulation of mRNAs for adrenal cytochrome P450 oxidases.

Adrenal Glands↗
PubMed1991

Heat shock inhibits the cytotoxic action of TNF-alpha in tumor cells but does not alter its noncytotoxic actions in endothelial and adrenal cells.

We have previously demonstrated that a short heat treatment protects target cells from lysis by tumor necrosis factors (TNFs). Here we show that a similar heat treatment of human umbilical vein endothelial cells and human fetal adrenal cells does not alter noncytotoxic actions of TNF, suggesting that heat shock may specifically inhibit the cytotoxic action of TNF. To find clues to the mechanisms by which heat shock protects cells from TNF killing, its effects on TNF-alpha-TNF-receptor interactions, on the metabolism of the ligand, and on the expression of mRNAs for possible protective proteins were studied. The affinity of binding and the internalization of the ligand were slightly reduced after heat shock. These effects were, however, very vague and seen both in heat-responsive tumor cells and in endothelial and adrenal cells. Thus, it is unlikely that they could explain the heat-induced TNF resistance. Heat shock increased the expression of mRNAs for heat shock proteins (hsps) 27 and 70 in all the cells studied, but did not alter the expression of manganous superoxide dismutase (MnSOD) mRNA, which has previously been shown to play a crucial role in TNF resistance. Based on these results, we suggest that cells have multiple mechanisms to escape TNF-mediated lysis and that heat-induced protection from TNF killing may be mediated by hsps or other heat-inducible protective proteins, which act after receptor binding and protect cells from TNF-induced cellular damage without inhibiting the signal transduction mediating noncytotoxic effects of TNF.

Adrenal Glands↗
PubMed1990

Pigmented villonodular synovitis and plasmacytoid lymphoma in a dog.

A 4-year-old Labrador Retriever was examined because of progressive left hind limb lameness involving the stifle. A villous synovial mass was evacuated by synovectomy. Initially, the macroscopic and histopathologic features suggested a malignant fibrosarcomatous process; however, further histologic studies revealed lesions consistent with pigmented villonodular synovitis. Nine months later, the dog developed a large retroperitoneal tumor, with metastasis to the lungs and liver. The dog was then euthanatized. By histologic and electron microscopic examinations, the tumor was found to be a primitive plasmacytoid lymphoma.

Animals↗
PubMed1990

Regulation of ACTH-induced steroidogenesis in human fetal adrenals by rTNF-alpha.

The presence of tumor necrosis factor type alpha (TNF-alpha) in different fetal tissue and adult adrenal extracts was investigated by radioimmunoassay (RIA). Measurable levels of TNF-alpha were found in 12/22 fetal adrenals, but in none of the seven adult adrenals studied. Since it is known that (i) steroidogenesis in fetal adrenals differs greatly from that in adult glands by having higher androgen/corticosteroid ratio, (ii) and that macrophage-derived factors may cause adrenocortical suppression, the effect of TNF-alpha on corticotropin-induced steroidogenesis in primary cultures of human fetal adrenals was studied. Results show that TNF-alpha effectively suppresses the production of cortisol and shifts the steroid synthesis towards androgen production. The effect was not accompanied by any change in cell viability and could be neutralized by addition of polyclonal rabbit anti-TNF-alpha antiserum to cell cultures. These results suggest that TNF-alpha may take part in the regulation of human fetal steroidogenesis within the network of the fetoplacental unit via inhibition of the cortisol synthesis.

Adrenal Glands↗
PubMed1990

Reduction by OK-432 of the monolayer contact-mediated inhibition of human natural killer cell activity.

In the present study we investigated the effect of OK-432, a streptococcus preparation, on the contact-mediated inhibition of human NK activity by primary cultures of monolayer cells. Either peripheral blood lymphocytes (PBL) or large granular lymphocytes (LGL) were incubated (2 x 10(6) cells/ml, total volume 2 ml) on confluent monolayer cells (uvea-derived fibroblasts, uvea-derived melanoma cells, or renal carcinoma cells) for 18 h in 24-well plates, washed twice, and tested for cytotoxicity against K562, a human myelogenous leukemia cell line, in a 4 h 51Cr-release assay. After contact with monolayer cells, NK activity of both PBL and LGL was significantly reduced. When these effector cells were preincubated with 0.1 U/ml of OK-432 for 18 h and then tested for the sensitivity to contact-mediated inhibition, the inhibition was significantly reduced. The pretreatment of monolayer cells with OK-432 or the addition of OK-432 into the coculture wells (of effector cells and monolayer cells) also significantly reduced the contact-mediated inhibition. Moreover, OK-432 (0.1 U/ml) reestablished the inhibited NK activity of PBL. These results suggest that OK-432 might enable NK cells to escape from the contact-mediated inhibition by monolayer cells and thus provide an additional potential mechanism for the observed clinical effectiveness of OK-432 reported by many groups.

Adenosine Triphosphate↗
PubMed1990

Swainsonine, an inhibitor of glycoprotein processing, enhances cytotoxicity of large granular lymphocytes.

In the present study we investigated the effects of inhibitors of glycoprotein processing on cytotoxicity of human large granular lymphocytes (LGL). The incubation of LGL for 36 h with 0.5 microgram/ml swainsonine (SW), which is an inhibitor of mannosidase II, resulted in the augmentation of cytotoxicity of LGL against an NK-resistant colon carcinoma cell line (Colo-320DM) without increase of binding frequency of LGL to target cells or of cell proliferation. The enhanced cytotoxicity was associated with increased binding of concanavalin A to SW-treated LGL. The augmentation of cytotoxicity was also seen by 1-deoxymannojirimycin (1-DMN), an inhibitor of mannosidase I, but much higher amounts of this agent were needed to get the same level of augmentation as that with SW. Other inhibitors of glycoprotein processing such as castanospermine and 1-deoxynojirimycin (1-DN) did not show any augmentative effects on LGL cytotoxicity. The enhancement of cytotoxicity by SW was abolished by the addition of rabbit anti-human interleukin (IL-2) antibody to the culture. This result suggests that IL-2 is involved in the augmentation of cytotoxicity of LGL by SW. The presence of SW in the culture of LGL together with IL-2 also enhanced LAK generation compared to that with IL-2 alone. Thus, our results suggest that SW should be recognized as an efficient immunopotentiator and that modulation of carbohydrate moieties elicited by SW may shed further light on the mechanism of LGL activation.

Alkaloids↗
PubMed1990

Interleukin-2 activated lymphocytes (LAK cells) as potential tumor tracers.

The homing characteristics and infiltrative capacity of interleukin-2 activated human peripheral blood lymphocytes, the lymphokine activated killer (LAK) cells, were studied. In vitro stimulated 111In-oxine labeled lymphocytes were injected into the hypogastric artery during hysterectomy, performed because of endometrial carcinoma. Scintigrams demonstrated clear homing of the lymphocytes into the area of the malignant tumor. No selective homing was detectable when labeled red blood cells were injected in a similar fashion. To analyze the infiltrative capacity of the activated lymphocytes, they were incubated in vitro with tumor spheroids grown from cultured glioma cell lines. As revealed by antibodies against the leukocyte common antigen and immunoperoxidase techniques, the activated lymphocytes infiltrated the three-dimensional tumor tissue slowly as a frontier. These results show that, in addition to their previously suggested potential role in cancer therapy, interleukin-2 activated lymphocytes may possibly also be useful as tumor tracers.

Female↗
PubMed1989

Heat shock protects WEHI-164 target cells from the cytolysis by tumor necrosis factors alpha and beta.

Elevated temperatures and a number of other types of stress induce synthesis of a small number of highly conserved proteins, the heat shock proteins, in a wide variety of cells. The structure and regulation of these proteins have been intensively studied but the question of the function of this universal response has remained unanswered. We studied the effect of heat shock on tumor necrosis factor-alpha (TNF-alpha)- and -beta (TNF-beta)-mediated cytolysis of WEHI-164 clone 13 target cells. One hour pretreatment of target cells at 42 degrees C decreased rTNF-alpha-mediated lysis by 65.3%, 50.5% and 44.8% and TNF-beta-mediated lysis by 61.9%, 43.2% and 38.9% at cytokine concentrations of 0.5 ng/ml, 5 ng/ml and 50 ng/ml, respectively, in an 18-h Cr-release assay. The effect was maximal when TNF-alpha was added 1 h after the heat shock and then gradually declined, being almost undetectable after 2 days. This pattern was found to roughly coincide with the kinetics of hsp68, the major heat-induced protein in murine cells. Heat shock treatment had no protective effect when given 1 h after addition of recombinant TNF-alpha. The heat-induced target cell resistance was not associated with decreased binding of recombinant TNF-alpha to its receptor. Inhibition of protein synthesis by cycloheximide diminished this effect by 76% and inhibition of transcription by actinomycin D abolished it completely, suggesting that de novo synthesized heat-induced proteins protect target cells from TNF-mediated lysis in heat shock-treated WEHI cells.

Animals↗
PubMed1989

Suppression of human natural killer cell activity by amino sugars.

In the present study we investigated the effect of amino sugars on human natural killer (NK) activity against K562, a human myeloid leukemia cell line, and Molt-4, a human T lymphoma cell line. The presence of amino sugars such as D-mannosamine, D-galactosamine, and D-glucosamine [6-25 mM (in the case of D-mannosamine, 1.5-12.5 mM)] in a 4-hr chromium-51 (Cr) release assay significantly inhibited NK activity of large granular lymphocytes (LGL) without affecting effector cell viability or spontaneous release from target cells. Sugars with acetylated amino residues (N-acetyl-D-mannosamine, N-acetyl-D-galactosamine, and N-acetyl-D-glucosamine) showed much smaller NK inhibition. Among the amino sugars tested, D-mannosamine was the strongest suppressor. When either LGL or K562 cells were pretreated with amino sugars and used in the 4-hr 51Cr release assay, only the pretreatment of effector cells resulted in the reduction of NK activity. The binding capacity of LGL to K562 cells, determined by a conjugate assay, was not reduced by the amino sugars enough to explain the strong inhibition of NK activity by these amino sugars, although some inhibitory effect on the binding of LGL to K562 cells was observed in some cases. In contrast, the polarization of the effector cell cytoskeleton, one of the energy-dependent steps, was significantly impaired. The cellular ATP level of LGL was also significantly reduced and the reduction of cellular ATP correlated well with the degree of the inhibition of NK cytotoxicity. These results suggest that the suppression of NK activity by amino sugars is due to the reduction of the ATP-based energy supply of the effector cells and that amino sugars, especially D-mannosamine, should be recognized as potent suppressors of natural cell-mediated immunity.

Adenosine Triphosphate↗
PubMed1989

N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) serine esterase in human cytolytic effector cells and cell line targets.

The granules of in vitro primed cytotoxic mouse T cells and cytotoxic cell lines have been shown to contain high levels of N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) esterase. The enzyme activity has been suggested to be associated with the cytotoxic capacity of killer cells. We investigated human leucocytes and found that neutrophils, monocytes, cytotoxic T lymphocytes (CTL), natural killer (NK) cells [large granular lymphocytes (LGL)], and interleukin 2 activated killer (LAK) cells, which all display efficient cytotoxic capacity, show only marginal BLT esterase activity. The low BLT esterase activity in human lymphocytes increases about twofold when cells are stimulated in vitro with interleukin 2 (IL-2), phytohaemagglutinin (PHA), or cultured in mixed lymphocyte culture (MLC). Mouse T lymphocytes have about 20 times more BLT esterase activity than human T lymphocytes. The BLT activity in mouse T cells also increases about twofold in MLC. The human leukaemia cell lines (K562, U937, MOLT-4, Jurkat) and the mouse mastocytoma line (P815), which are frequently used as target cells, contain more BLT esterase activity than human resting or activated lymphocytes. We did not find a direct correlation between the cytotoxic capacity and the BLT esterase activity of killer cells.

Animals↗
PubMed1989

Tumor necrosis factor in the human fetoplacentary unit.

In the present paper we review our findings on tumor necrosis factor (TNF) in human placental and adrenal tissues and suggest a possible novel biological role for this multi-functional lymphokine. TNF was found in the second and third trimester amniotic fluids in concentrations varying from 1.7 to 2.5 ng/ml on the average. In the amniotic fluid TNF was present in a biologically inactive form but in freshly-prepared supernatants of placental and decidual tissue homogenates corresponding levels were also found with bioassays based on the cytotoxicity of TNF to WEHI cells. In screening other fetal tissues we could detect biologically active TNF also in homogenates of fetal adrenals, and cultured fetal adrenal cells produced TNF upon stimulation with ACTH. A possible biological function for TNF was suggested by the findings that is suppressed the ACTH stimulated cortisol synthesis of fetal adrenal cultures and switched the steroidogenesis towards dehydroepiandrosterone (DHEA) and its sulphate (DHEAS). These are important precursors for placental steroid synthesis which are mainly provided by the fetal adrenals during normal gestation, and the results thus suggest a role for TNF in the regulation of steroidogenesis in the human fetoplacentary unit.

Adrenal Glands↗
PubMed1988

Cyst fluid of glioma does not inhibit the killing action of lymphokine-activated killer (LAK) cells in vitro.

In theory, lymphokine-activated killer (LAK) cells offer a potential method to treat cerebral gliomas, especially low-grade gliomas. LAK cells would be administered by repeated injections straight into the cavity of a subtotally removed tumour. However, brain-tumour cyst fluid has been shown to be immunosuppressive in lymphocyte stimulation tests. Therefore we wanted to know whether the fluid would reduce the killing efficacy of LAK cells. Using a standard cytotoxity test based on 51Cr release, we compared in vitro the cytotoxity of LAK cells against K-562 tumour cells in brain-tumour cyst fluid, autologous serum and allogeneic serum. Five patients with cystic glioma and one with cystic meningioma were studied and no inhibition of cytotoxity of LAK cells was observed.

Adolescent↗
PubMed1988

Characteristics of soluble tumour-derived proteins that inhibit natural killer activity.

We have previously shown that various benign and malignant natural killer (NK)-resistant monolayer cells inhibit endogenous human NK activity, probably by reducing the secretion of cytotoxic factors from the effector cells. The nature of the molecules responsible for the inhibition has been unclear. In this study we show that phosphate-buffered saline (PBS) extracts of ovarian cystadenocarcinoma tissue and normal uterine smooth muscle strongly inhibit NK activity. Fractionation of tumour extracts by gel chromatography revealed major inhibitory activity in the Mr range 160,000-180,000, and other weaker inhibiting activities in the Mr ranges 50,000-70,000 and 20,000. The active material of Mr range 160,000-180,000 was adsorbed on anion exchange chromatography column at neutral pH and physiologic NaCl concentration, and it was eluted by 0.31-0.34 M NaCl. The inhibitory molecule was sensitive to proteolysis. No relation of this compound to immunoglobulins or trypsin and urokinase inhibitors was detected. The unfractionated extract inhibited NK activity apparently by the same mechanism as the monolayer target cells, i.e. by reducing the secretory capacity of effector cells. The data strongly suggest that the NK-inhibiting compounds described in this work are involved in the inactivation of NK cells by intact monolayer cells.

Cytotoxicity, Immunologic↗
PubMed1988

Demonstration of tumor necrosis factor in human amniotic fluids and supernatants of placental and decidual tissues.

Human amniotic fluid samples obtained from normal pregnancies and supernatants of normal full-term placental and decidual tissues were assayed in a specific radioimmunoassay (RIA) and in a sensitive bioassay for tumor necrosis factor (TNF). TNF was detected in 91% of amniotic fluid samples and in all placental and decidual supernatants assayed by the RIA. Amniotic fluid samples collected during the second trimester of pregnancy contained significantly higher concentrations of TNF than samples collected during the third trimester, average concentrations being 2.5 ng/ml and 0.9 ng/ml, respectively. Parallel testing in the bioassay showed that most of the amniotic fluid TNF is biologically inactive, whereas placental and decidual supernatants had biologic activity correlating with TNF levels detected by the RIA. The quantities of TNF detected by the RIA ranged from 1.1 to 2.8 ng/ml in placental supernatants and from 3.9 to 8.5 ng/ml in decidual supernatants. The presence of TNF in normal amniotic fluids and in placental and decidual supernatants suggest that this cytokine has a physiologic role in human pregnancy.

Amniotic Fluid↗
PubMed1987

Interleukin-2-stimulated natural killer activity against malignant and benign endometrium.

Natural cell-mediated immunity against autologous tumor cells, autologous endometrial epithelium, and allogeneic epidermoid carcinoma cell line HeLa was tested in 8 patients with endometrial carcinoma and one patient with endometrial stromal sarcoma. The average cytotoxicity of unstimulated peripheral blood lymphocytes against autologous tumor and HeLa cells was weak but significant. Pretreatment of effector cells for 3-5 days with 300 U/ml recombinant interleukin-2 (rIL-2) resulted in increased cytotoxicity against malignant target cells in 7 out of 9 cases. The 2 patients' effector cells which were refractory to rIL-2 could be stimulated to appreciable lytic activity against the malignant target cells with a recently described cytokine which induces morphological differentiation of natural killer cells. Benign endometrial cells were weakly sensitive to rIL-2-activated lysis in 2 cases. The precursors of the rIL-2-activated killer cells were mostly CD16-positive and CD3-negative, and co-sedimented with endogenous natural killer cells in discontinuous density gradient centrifugations. These results indicate the rIL-2-activated killer cells have a capacity to distinguish between normal and malignant endometrial cells, and that the precursors of the lytic cells in this system belong to the same subpopulation of lymphocytes as endogenous natural killer cells. In addition, rIL-2 alone may not in all cases be sufficient for optimal generation of cytotoxicity against malignant cells.

Adult↗
PubMed1987

Mechanism of cell contact-mediated inhibition of natural killer activity.

Natural killer cell activity is inhibited by primary cultures of monolayer cells. In this study, we analyzed the mechanism of the inhibition. Inhibited NK cells showed unaltered binding capacity to NK sensitive K562 cells. The orientation of the effector cells' actin-containing microfilaments, an event known to occur during the programming for the lysis stage in lytic conjugates, was unaffected by the inhibition. In single cell cytotoxicity experiments, the number of killer cells among conjugate-forming cells was reduced. The capacity of the inactivated NK cells to secrete cytotoxic factors upon stimulation with Con A was also impaired. Both NK-resistant inactivating target cells and NK-sensitive K562 cells were sensitive to the toxic factors secreted by NK cells. Thus, the results indicate that the target cell-mediated inactivation of NK cell is based on a block in the lethal hit stage, possibly due to reduced release of toxic factor(s) from the effector cells. The capacity of inactivated effector cells to mediate antibody-dependent cellular cytotoxicity was unimpaired, suggesting that the contact-mediated inhibition of cytotoxicity selectively affects NK cells.

Antibody-Dependent Cell Cytotoxicity↗