Reduced expression of cellularfos gene in peripheral blood mononuclear cells of patients with primary biliary cirrhosis

Abnormal expression of certain cellular oncogenes in peripheral blood mononuclear cells (PBMC) is associated with several autoimmune disorders and is thought to reflect a pathologically activated state in various lymphocytic subpopulations. Using the Northern blot hybridization technique, we studied the expression of cellular (c-)fos, c-myc and N-ras genes in PBMC isolated from patients with primary biliary cirrhosis (PBC) and normal control subjects. The expression of c-fos gene was reduced significantly in patients with PBC, while there was no significant difference between the two groups in the expression of c-myc and N-ras genes. Stimulation of PBMC with mitogens in vitro increased c-fos gene expression markedly to a similar extent in both normal subjects and in patients with PBC. Furthermore, the reduced expression of c-fos gene recovered significantly in all the patients examined after treatment with ursodeoxycholic acid (UDCA). These results suggest that the reduced expression of c-fos gene in PBMC is a reversible functional manifestation of PBC and that UDCA therapy for PBC may improve abnormal lymphocyte function.     

Expression of myc and ras oncogenes in two newly established neuroblastoma cell lines

Summary Two new neuroblastoma cell lines, KG-MH and KM-YH have been established from fresh tumour samples. In vitro growth characteristics are presented together with a karyological analysis. Northern and Southern blot experiments have been performed using molecularly cloned probes for c-myc, N-myc, c-Ha-ras, c-Ki-ras, and N-ras oncogenes. Both cell lines showed expression for N-myc, while c-myc expression was not detected. Cell line KM-YH, with a rather long population doubling time of 78 h, showed additional expression for the three ras genes.     

ADHESION OF RHEUMATOID LYMPHOCYTES TO MUCOSAL ENDOTHELIUM: THE GUT REVISITED

By a study of the adhesion of rheumatoid mononuclear cells,we have sought to clarify the homing properties and originsof cells likely to be involved in the pathogenesis of this disease.The adhesion of mononuclear cells from patients with rheumatoidarthritis (RA) was enumerated by an in vitro adherence assayusing frozen sections of endothelium-containing gut lamina propria(EGLP) from porcine small intestine. Preliminary studies verifiedthe involvement of known adhesion molecules by inhibition assaysusing monoclonal antibodies Meca-367, Mel-14 and Hermes-3. Twenty-fivepaired samples of peripheral blood (PB) and synovial fluid (SF)were studied, plus six from synovial membrane (SM) and eightfrom patients with other diseases. There was a significantlygreater degree of adherence to EGLP by SF cells than PB (meanadherence 266 ± 22 cells/mm², compared to 136 ±13 cells/mm², respectively, the majority of which were CD8+cells; P = 0.02, Mann-Whitney U-test for 25 paired samples).The results of the monoclonal antibody inhibition assays werein keeping with the involvement of homing receptors to gut endotheliumin our assay system. Synovial fluid lymphocytes from RA patientsexhibited adhesion properties more in keeping with lymphocytesof mucosal than of lymph node origin. Synovial membrane lymphocytes,by contrast, showed poor adherence to endothelium-containinglamina propria. The gut, as an immune lymphoid organ, may thusplay a contributory role in this disease, possibly through thepathological seeding of cells into the synovial space. KEY WORDS: Rheumatoid arthritis, Adhesion, MALT     

Adrenergic agents,but not triiodo-L-thyronine induce c-fos and c-myc expression in the rat heart

Summary We have examined the expression of two nuclear-acting oncogenes, c-fos and c-myc in the rat heart following administration of hormones implicated in the development of cardiac hypertrophy. A single injection of norepinephrine (2.5 g/kg to 2.5 mg/kg) led to transient increases in the levels of both c-fos and c-myc mRNA. The response was sequential: elevated levels of c-fos mRNA were first observed 15 min after treatment and peaked at 1 h whilst c-myc mRNA levels increased 30 min after treatment and peaked at 2h. The response of both cellular oncogenes to norepinephrine was reduced significantly by a blockade but (3 blockade was less effective. Administration of triiodo-L-thyronine (0.25 mg/kg), a level known to promote cardiac hypertrophy, did not produce elevated levels of c-fos or c-myc mRNA. In an initial study, it was possible to demonstrate induction of c-fos and c-myc in rat hearts perfused in vitro with medium containing 2x10^–7 M norepinephrine. These results provide support for the notion that c-fos and c-myc expression may play a transducing role in the development of adrenergic-mediated, but not thyroid hormone-mediated cardiac hypertrophy.     

Association of genetic alterations of c-myc,c-fos,and c-Ha-ras proto-oncogenes in colorectal tumors

Using Northern and dot-blot analysis we examined normal and tumor tissue from 29 patients with colorectal carcinomas for the expression and amplification of c- myc,c-fos and c-Ha-ras proto-oncogenes. Overexpression of c-myc (6/24), c-fos (4/24), and c-Ha- ras (9/23) was found. For the c- fos proto-oncogene we also have observed decreased levels of expression in 13 percent (3/24) of the cases analyzed. Gene amplification appeared to be a rare event in these tumors and was found in 3/29 (10 percent) tumors for c- myc and in 1/29 (3 percent) for c- fos protooncogene.Curves for overall survival and for disease-free survival failed to show a significant tendency in these parameters to be poorer in tumors with alterations of gene expression for any of the proto-oncogenes analyzed. Despite the biologic importance of these genetic alterations in the etiology of colorectal tumors, levels of c- myc,c- fos,and c-Ha- ras gene expression separately or together cannot be considered as prognostic factors for clinical outcome of the disease.This work was supported by grant 507750/87 of the Conselho Nacional de Desenvolvimento Cientificoe Tecnologico.     

EXPRESSION OF bcl-2 IN RHEUMATOID ARTHRITIS

Since defective apoptosis has been suggested to play a rolein the development of autoimmune diseases, we have investigatedthe expression of the proto-oncogene bcl-2 in patients withrheumatoid arthritis (RA). The expression of bcl-2 was studiedin peripheral blood (PB) and synovial fluid (SF) lymphocytesand synovial tissues (ST) from patients with RA using immunohistochemistry,flow cytometry and nucleic acid hybridization. Patients withreactive arthritis (ReA) or osteoarthritis (OA) and healthyindividuals were used as controls. The expression of bcl-2 proteinin PB lymphocytes and the expression of bcl-2 mRNA in PB mononuclearcells (PBMC) was similar in healthy controls and patients withRA. However, bcl-2 protein expression was significantly reducedin SF lymphocytes when compared to PB lymphocytes. Similar resultswere observed with lymphocytes from patients with ReA, and irrespectiveof whether total lymphocytes, T cells or different T-cell subsetswere studied. In the synovial sections, the expression of bcl-2was restricted to lymphocytes, and bcl-2+ cells were observedin the majority of samples from patients with RA, OA and ReA.These data indicate that the expression of bcl-2 is not increasedin the lymphocytes or ST derived from patients with RA. Instead,decreased expression of bcl-2 protein in SF lymphocytes comparedto PB lymphocytes was demonstrated. We suggest that bcl-2 doesnot play a significant role in the pathogenesis of RA. KEY WORDS: bcl-2, Rheumatoid athritis, Lymphocytes     

Varied expression of major histocompatibility complex and oncogenes in shope carcinoma cell lines derived from a single tumor

The cellular gene expression was compared in four Shope carcinoma cell lines, which were derived from a single tumor and possess various potentials for differentiation and tumorigenicity. The E6 and E7 transforming genes of cottontail rabbit papillomavirus were expressed in all these cell lines, highest level of expression being in the most tumorigenic and undifferentiated cell line, where the major histocompatibility complex (MHC) class I expression was the lowest. The MHC class II antigen, which is not expressed on normal epithelial cells, was detected in all the cell lines, but hardly, if at all, on the surface of these cells. The surface expression of the MHC class II antigen could not be induced by the culture supernatant of phytohaemagglutinin-stimulated splenocytes, which increased the surface expression level of the MHC class I antigen of the same cells. These findings suggest that the aberrant expression of the MHC class II antigen in these cells could not be implicated in the immune response against tumors. The c-fos, c-myc and c-H-ras oncogenes were variably expressed in these cell lines, but there was no correlation with tumorigenicity.     

Effects of retinoic acid on NIH3T3 cell transformation by the H-ras oncogene

Summary Exposure of NIH3T3 cells to retinoic acid resulted in a dose-dependent modulation of transformed focus formation after transfection with an activated H-ras oncogene. Inhibition induced by 10 M retinoic acid was maximal at 21.4% of control values. Maximal inhibition of transformation was found after exposure to 10 M retinoic acid between days 0 and 3 of the transfection period. This concentration was also inhibitory for colony formation upon transfection of the non-transforming geneaph, suggesting that retinoic acid acts primarily on the process of transfection to inhibit focus or colony formation. Exposure to retinoic acid during the late period of the transfection protocol (days 14–20) resulted in alterations in focus morphology. A transformed cell line containing H-ras underwent reversion of the transformed phenotype after 4 weeks of treatment with retinoic acid, as determined by alterations in cell morphology and anchorage-independent growth. Phenotypic reversion was not associated with changes in the expression of the exogenous H-ras or endogenous c-myc or c-fos oncogenes.Abbreviations DMEM Dulbecco's modified Eagle medium - SSC saline sodium citrate - TGF transforming growth factor This investigation was supported by grants CA 52925, CA 13343 and ES 00260     

Relationship of plasma folic acid and status of DNA methylation in human gastric cancer

To evaluate the anti-cancer effects of folic acid at the molecular level, we determined plasma folic acid concentration by radioimmuno-assay and the degree of total genomic DNA methylation by incubating DNA with³H-S-adenosylmethionine (³H-SAM) in the presence of a methylase, and analyzed the methylation status of the c-myc and c-Ha-ras oncogenes by Southern blotting in 21 patients with advanced gastric cancer. The degree of total genomic DNA methylation of cancerous tissues was significantly lower than that of paracancerous and non-cancerous tissues; c-myc and c-Ha-ras oncogenes from cancerous (10/21, 5/10) and paracancerous (13/21, 4/10) tissues were hypomethylated. The plasma folic acid concentration in patients who showed hypomethylation was lower than that patients showing normal methylation. These findings suggest that a decrease in folic acid, and the subsequent DNA hypomethylation, may be involved in human gastric carcinogenesis.     

A QUANTITATIVE STUDY OF THE PHAGOCYTOSIS OF URATE CRYSTALS IN THE SYNOVIAL FLUID OF ASYMPTOMATIC JOINTS OF PATIENTS WITH GOUT

The objective of this study was to-determine whether monosodiumurate (MSU) crystals are phagocytosed in the synovial fluid(SF) of the asymptomatic joints of patients with gout. SF sampleswere obtained from 20 asymptomatic knees of 19 different patients.Cell and differential counts were done. Intracellular MSU crystalswere identified by ordinary and polarizing light microscopy.We found that in 19 out of the 20 SF samples intracellular MSUcrystals have been found. A mean of 22.55% [confidence interval(CI) 13.92, 31.18; range 0–62] of all the cells containedintracellular MSU crystals. The majority of the cells whichcontained intracellular crystals were mononuclear cells (MC)and polymorphonuclear (PMN) leucocytes containing intracellularcrystals accounted only for 0.5% (CI 0, 1.05; range 0–5)of the total. The total cell count was 527 cells/mm³ (CI 226,828, range: 30–2670). Poor correlation was found betweenthe percentage of cells with intracellular crystals and boththe total cell count (r = –0.22) and the percentage ofPMN leucocytes (r = –0.26). We conclude that cells containingphagocytosed MSU crystals—generally mononuclear cells—area regular finding in the SF of asymptomatic joints of patientswith gout. This finding indicates that other factors besidesintra-articular interaction between crystals and cells are necessaryto produce arthritis in gouty patients. KEY WORDS: Crystal arthritis, Gout, Joint fluid     

T lymphocytes in the synovial fluid of patients with active rheumatoid arthritis display CD134-OX40 surface antigen.

OBJECTIVE: To assess the percentage of T lymphocytes, bearing CD134, a member of the TNF receptor superfamily, primarily found on autoreactive CD4+ T cells in the peripheral blood (PB) and synovial fluid (SF) of rheumatoid arthritis (RA) patients. METHODS: The surface expression of CD134 on SF and PB mononuclear cells was performed by flow cytometry in 25 RA patients and correlated to the disease activity. RESULTS: CD134 expression on CD3+, CD4+, CD8+ and CD25+ cells was higher in SF than in PB of RA patients (P < 0.001). No differences were observed in the percentage of CD134+/CD4+ T lymphocytes in the PB of RA patients and controls. Patients with active RA had significantly higher percentage of CD3+/CD134+, CD4+/CD 134+, CD8+/CD134+ and CD25+/CD 134+ than those with inactive disease. CONCLUSION: These findings suggest that CD134+ T cells are involved in the immunopathological process of RA synovitis, maybe mirroring some other autoimmune disease in which autoreactive T cell infiltrating the target tissues largely coexpress CD134.     

Modulation of spontaneous immunoglobulin production by natural killer cells in rheumatoid arthritis

Synovial fluid (SF) mononuclear cells obtained from patients with rheumatoid arthritis (RA) spontaneously produce large amounts of immunoglobulin. In the rheumatoid joint, natural killer (NK) cell activity is reduced in comparison with that in the peripheral blood (PB). We examined the ability of SF NK cells to modulate the spontaneous production of Ig in RA SF, and we contrasted this with the activity in PB from RA patients and from normal subjects. We found that the spontaneous production of IgG was greater in RA SF than in RA or normal PB. The baseline NK activity was significantly lower in RA SF than in RA or normal PB (P less than 0.005). Incubation with anti-Leu-11b and complement reduced NK activity in PB, but not in SF, and it significantly (P less than or equal to 0.021) increased IgG production in both RA SF and RA PB. Lysis of NK cells in this manner also resulted in a significant increase (P less than 0.02) in IgM production in RA SF. These results suggest that NK cells with a Leu-11b phenotype down-regulate the ongoing synthesis of IgG and IgM in the rheumatoid joint.     

Phenotypes of T lymphocytes from peripheral blood and synovial fluid of patients with rheumatoid arthritis and juvenile rheumatoid arthritis. Evidence in favour of normal helper and suppressor functions of T lymphocytes from patients with juvenile rheumatoid arthritis

Lymphocytes from peripheral blood (PB) and synovial fluid (SF) from 21 patients with rheumatoid arthritis (RA) and 18 patients with juvenile rheumatoid arthritis (JRA) were studied with respect to T cell phenotypes using monoclonal antibodies in a rosette assay. The percentage of HLA-DR positive T cells was counted in PB and SF using indirect immunofluorescence. Suppressor cell activity of T cells from PB and SF was investigated by measuring the immunoglobulin production by pokeweed mitogen (PWM) stimulated B cells mixed with T cells at various ratios. The mean T4/T8 ratio was significantly lower in SF than in PB of both RA and JRA patients (p = 0.0062 and p less than 0.0001 respectively). The mean percentages of HLA-DR positive T cells were elevated in SF compared with PB in both patients groups (p less than 0.03 and p less than 0.04 in RA and JRA patients respectively). Mean suppressor cell activity and helper cell activity of T cells from SF and PB of JRA patients was normal. Thus there seems to be a dichotomy between the number of T8+ cells and suppressor cell function in mononuclear cells from SF of patients with JRA. This indicates that a considerable proportion of the T8+ cells in the SF do not have suppressor functions.     

Expression of L-Selectin,CD43, and CD44 in Synovial Fluid Neutrophils from Patients with Inflammatory Joint Diseases

Objective. To study the expression of L-selectin, CD43, and CD44 on peripheral blood (PB) and synovial fluid (SF) neutrophils from patients with inflammatory joint diseases, and to investigate the presence of soluble L-selectin in both SF and plasma from patients with acute and chronic arthritis. Methods. PB and SF neutrophils were isolated from 13 patients with rheumatoid arthritis (RA) and 17 patients with various inflammatory joint diseases other than RA. Expression of L-selectin, CD43, CD44, CD11a, and CD11b was determined in both unstimulated and in vitro—activated cells by immunofluorescence flow cytometry. Soluble L-selectin levels were estimated in SF and plasma by a semiquantitative radioimmunoassay. Results. Neutrophils from SF showed diminished expression of L-selectin compared with PB neutrophils; CD43 expression and CD44 expression were decreased in SF neutrophils from most patients. In contrast, SF neutrophils exhibited significantly increased expression of CD11b, to an extent similar to that seen with in vitro—activated PB neutrophils. Soluble L-selectin was detected at similar levels in SF and PB. Conclusion. The phenotypic profile of SF neutrophils (low levels of L-selectin, CD43, and CD44, and high levels of CD11b) from most patients with RA or other inflammatory joint conditions resembles that observed in in vitro—activated neutrophils. Our results suggest that SF neutrophils are activated to a similar degree in inflammatory joint diseases with different pathogenic mechanisms.     

Overexpression of BP1, a homeobox gene,is associated with resistance to all-trans retinoic acid in acute promyelocytic leukemia cells

BP1, a homeobox gene, is overexpressed in the bone marrow of 63% of acute myeloid leukemia patients. In this study, we compared the growth-inhibitory and cyto-differentiating activities of all-trans retinoic acid (ATRA) in NB4 (ATRA-responsive) and R4 (ATRA-resistant) acute promyelocytic leukemia (APL) cells relative to BP1 levels. Expression of two oncogenes, bcl-2 and c-myc, was also assessed. NB4 and R4 cells express BP1, bcl-2, and c-myc; the expression of all three genes was repressed after ATRA treatment of NB4 cells but not R4 cells. To determine whether BP1 overexpression affects sensitivity to ATRA, NB4 cells were transfected with a BP1-expressing plasmid and treated with ATRA. In cells overexpressing BP1: (1) proliferation was no longer inhibited; (2) differentiation was reduced two- to threefold; (3) c-myc was no longer repressed. These and other data suggest that BP1 may regulate bcl-2 and c-myc expression. Clinically, BP1 levels were elevated in all pretreatment APL patients tested, while BP1 expression was decreased in 91% of patients after combined ATRA and chemotherapy treatment. Two patients underwent disease relapse during follow-up; one patient exhibited a 42-fold increase in BP1 expression, while the other showed no change. This suggests that BP1 may be part of a pathway involved in resistance to therapy. Taken together, our data suggest that BP1 is a potential therapeutic target in APL. Rania T. Awwad and Khanh Do contributed equally to the present study.     

Restricted cytokine expression in rheumatoid arthritis

Objective. To determine the cytokine profile of the phenotypically activated T cell in rheumatoid arthritis (RA) synovium. Methods. Interleukin-2 (IL-2), IL-2 receptor (IL-2R), IL-6, IL-4, and interferon-γ (IFNγ) gene expression was examined in T cells from freshly isolated synovial fluids (SF) and synovial tissues (ST) from patients with RA. Estimates of baseline expression were determined using unstimulated peripheral blood (PB) T cells from healthy individuals. The corresponding positive controls were phytohemagglutinin-activated tonsil T cells. Results. In studies of paired PB and SF T cell samples from 17 RA patients, IL-2 messenger RNA (mRNA) levels in only 1 PB and 3 SF samples were more than 2 standard deviations above the mean of levels in unstimulated PB from healthy donors. Similarly, only 5 PB and 7 SF samples exhibited elevated IL-2R mRNA levels. IFNγ gene expression was not detected in any of the paired RA PB or SF samples. Fractionated T cells from 12 RA ST were screened with similar results: Only 1 of 12 samples exhibited IL-2 mRNA levels more than 2 standard deviations above levels in baseline controls. IL-2R mRNA levels were low or not detected, and IFNγ mRNA was absent. Subsequent studies showed that IL-4 and IL-6 gene expression levels were also low in RA tissues compared with tonsil T cell–positive controls. Conclusion. These data provide evidence for restricted cytokine expression in the T cell population in RA tissues.     

Decreased expression of c-myc oncoprotein by peripheral blood mononuclear cells in thalassaemia patients receiving desferrioxamine

Abstract: Desferrioxamine (DFX) is an iron chelation agent widely used in the treatment of transfusional iron overload in patients with thalassaemia major and other severe refractory anaemias. DFX has been shown to induce inhibition of DNA synthesis and apoptosis in vitro; however, the molecular targets of DFX action are not well known. The c-myc proto-oncogene is involved in a number of cellular processes including proliferation, differentiation and apoptotic cell death. We have examined the expression of c-myc in peripheral blood mononuclear cells from 71 patients with homozygous β-thalassaemia in regular transfusion and iron chelation therapy with DFX, 5 non-transfusion, non-chelation-dependent thalassaemic patients, and 15 healthy volunteers using an APAAP immunocytochemical method. We have found that mononuclear cells from thalassaemic patients receiving DFX express significantly lower levels of c-myc protein compared to control healthy volunteers or thalassaemics receiving no DFX. In vitro treatment of HL60 or K562 leukaemic cells with 100 μl DFX also induced a rapid decrease in c-myc mRNA and protein levels, followed by apoptosis and inhibition of DNA synthesis. These effects were blocked by simultaneous addition of ferric chloride. Our data suggest that deprivation of cellular iron induces downregulation of c-myc expression in vitro and in vivo and may influence haemopoietic cell growth and survival.