Biomedical subjects

M Zembala

Publications and source records attributed to M Zembala.

At least 37 records · Page 2Linked to original sources

PubMed2001

Three-color flow cytometry detection of intracellular cytokines in peripheral blood mononuclear cells: comparative analysis of phorbol myristate acetate-ionomycin and phytohemagglutinin stimulation.

The assessment of intracellular cytokines at the single-cell level by flow cytometry has recently become a potent tool in many areas of cell biology and in defining the role of cytokines in various human diseases. Three-color flow cytometry for detection of intracellular cytokines combined with simultaneous determination of lymphocytes (CD3(+) and CD4(+)) or monocytes (CD33(+) and CD14(+)) was used for comparison of phytohemagglutinin (PHA)-and phorbol myristate acetate (PMA)-ionomycin-induced production of intracellular cytokines in peripheral blood mononuclear cells (PBMCs) of healthy donors. We found that the number of PBMCs stained for tumor necrosis factor alpha and gamma interferon after 6 h of activation was higher when PMA-ionomycin was used for stimulation, while the frequencies of cells positive for interleukin 4 (IL-4) were similar for both stimulators. However, PMA-ionomycin stimulation caused prominent alterations of cell morphology and membrane expression of CD4 and CD14. In contrast, PHA did not cause downregulation of surface markers and resulted in less pronounced alterations in both forward and side scatter signals during flow cytometry analysis. Moreover, during 48 h of culture PHA stimulated tumor necrosis factor beta and IL-10 production, which was not observed when PMA-ionomycin was used. We conclude that the use of PHA for cell activation may limit in vitro artifacts and allow more precise analysis of intracellular cytokine production in various disease states.

Antigens, CD↗

PubMed2001

[Transcatheter closure of atrial septal defects in adults with the Amplatzer atrial septal occluder].

UNLABELLED: In many centres the Amplatzer Septal Occluder (ASO) (AGA Med. Corp. Minnesota, USA) has become the device of choice for secundum atrial septal defect (ASD) closure in children. Current trend towards transcatheter closure of ASD in children could be translated to adults and many patients (pts) may avoid the need of open heart surgery. Assessment the efficacy and complication of device occlusion of ASD in adults, using ASO. Between October 1997 and April 2001 transcatheter closure of ASD was attempted in 51 pts who fulfilled the inclusion criteria--significant shunt with sufficient rims of interatrial septum. Mean age of pts was 29 (16-63) y, mean ASD diameter assessed by transesophageal echocardiography (TEE) was 14.7 (7-24) mm, assessed during catheterization by balloon sizing (stretched diameter) was 20.2 (8-36) mm. There were 9 pts with multiple ASDs, 2 pts with aneurysm of interatrial septum and 2--after previous surgery (recanalization of ASD). The ASO devices were successfully implanted in all, but one pt. In one patient because of unstable position of ASO (floppy rims), device was removed and bigger one was applied during next session. In one case early embolization to abdominal aorta occurred, ASO was translocated to aortic arch with Dotter basket and removed from aorta during simultaneous surgical closure of ASD. Mean fluoroscopy time was 15 (4-50) min. The occlusion rate after 24 h was 90%, after 1 month (m) 92%, after 3 m 93.5%, after 1 year (y) 93.3% and after 2 y 93.3%. All residual shunts were trivial. There were no late complication. CONCLUSIONS: The excellent results of ASD closure with ASO in adults indicate this treatment as a method of choice in selected patients, but long term follow-up is necessary to state final judgement.

Adolescent↗

PubMed2000

The effect of pentoxifiline on post-injury hyperalgesia in rats and postoperative pain in patients.

Recent studies demonstrate that activation of proinflammatory cytokines following injury intensifies the process of nociception. The present investigation assessed the influence of pre-injury pentoxifiline (PTFL, a non-specific cytokine inhibitor) on the development of post-injury nociception in animals and patients. It was established that intrathecal or intraperitoneal PTFL, elevated the nociceptive threshold for mechanical stimuli in the formalin test in rats. Pre-injury PTFL also inhibited pain-related behaviour. These findings correlate with a lower TNFalpha level in the serum of animals receiving pre-injury PTFL. In clinical investigations PTFL was administered intravenously before elective cholecystectomy. Patients who received preoperative PTFL had lower opioid requirements in the early postoperative period than control. At the same time, serum levels of TNFalpha and IL6 were lower in the PTFL group. Our results confirm the hypothesis as to the possibility of modulating of nociception through preemptive administration of a cytokine inhibitor.

Adult↗

PubMed2000

[Results of kidney transplantation in Krakow in 1992-2000].

The aim of the study was an analysis of renal transplantation results in the Krakow Transplant Center during 1992-2000. The analysis concerned 94 cadaveric transplant recipients. The study group included 31 females aged 23 to 61 years (mean 40.4 years) and 63 males aged 16 to 60 years (mean 41.8 years). The time of pre-transplant renal replacement therapy ranged from 4 to 120 months (mean 32 months). The mean time of total ischaemia was 22 hours 20 minutes. The majority of the recipients had three identical antigens out of six typed. Most of the recipients were treated with three immunosuppressive drugs including: Cyclosporine A, Azathioprine and steroids. Immediately after kidney transplantation 25.6% of the patients had urine output and did not require dialysis. Acute renal failure (ARF) of the graft was observed in 73.2% recipients. The average number of hemodialysis sessions in patients presenting ARF was 10. Acute rejection was diagnosed in 41.5% of the patients. The most frequent complications were: CMV (cytomegalovirus) infection, UTI (urinary tract infection) and policytemia. In the study group 1-year survival rate of the patients was 97.8% and 1-year graft survival was 93.61%. The 5-year survival rates both in the patients and the grafts were very satisfactory (96.96% and 87.7% respectively).

Acute Kidney Injury↗

PubMed1999

Demonstration of iNOS-mRNA and iNOS in human monocytes stimulated with cancer cells in vitro.

Synthesis and localization of inducible nitric oxide synthase mRNA (iNOS-mRNA) and iNOS protein in the cultures of human monocytes (Mphi) and colon carcinoma cell line (DeTa) that resulted in nitric oxide (NO) synthesis has been studied. The iNOS-mRNA was observed around the sixth hour of culture and peaked at the twelfth hour. The iNOS-mRNA, as determined by the in situ hybridization and iNOS protein, as detected by staining with specific anti-iNOS monoclonal antibodies, were observed preferentially in the cytoplasm of some Mphi, but not in cancer cells. Mphi cultured alone did not show detectable iNOS-mRNA expression and iNOS protein. Mphi sorted out from tumor cells after 8 h of co-culture expressed iNOS protein and iNOS-mRNA, which were not detected in Mphi without previous contact with cancer cells. Prevention of NO synthesis by (L-N5-1-iminoethyl)-ornithine (L-NIO) partly inhibited Mphi cytotoxic activity against DeTa (NO-inducing cancer cell line) but not against the human pancreatic cancer (HPC-4) cell line that does not induce NO production in Mphi. This suggests that Mphi cytotoxic activity, at least in some cases, may be NO dependent. These observations provide further evidence that Mphi can be directly stimulated by cancer cells for de novo production of NO and suggest that iNOS occurring in the tumor-infiltrating macrophages may arise as a result of their interactions with tumor cells. However, because only some tumor cells are able to induce NO production in a small proportion of Mphi, its role in the anti-tumor response of the host is probably limited.

Adenocarcinoma↗

PubMed1999

Involvement of protein kinases in signalling for nitric oxide (NO) and tumour necrosis factor alpha (TNF) production by monocytes stimulated with colorectal DeTa cancer cells: the lack of evidence for the role of TNF in the regulation of NO production.

Kinases that are involved in NO and TNF production by human monocytes (MO) stimulated by colorectal cancer (DeTa) cells and effects of exogenous and endogenously synthesized TNF on NO induction were studied. The results based on the use of various inhibitors of protein kinases suggest that different signalling pathways operate in MO during induction of TNF and NO release after stimulation by DeTa cells. Stimulation of NO production required at least PTK, PKC and PKA, but only PTK and PKC were engaged in signal transduction for TNF production. Exogenous TNF and TNF produced by MO upon contact with DeTa cells was not sufficient for the induction or enhancement of NO synthesis in MO. The TNF synthesis was not influenced by neither exogenous nor endogenous NO produced by MO in the co-culture. Therefore, signal transduction pathways operating in MO during NO induction seem to be different from these engaged in TNF production, and both regulatory pathways probably operate in MO independently.

Carcinoma↗

PubMed1999

Induction of reactive oxygen intermediates in human monocytes by tumour cells and their role in spontaneous monocyte cytotoxicity.

The present study examined the ability of human monocytes to produce reactive oxygen intermediates after a contact with tumour cells. Monocytes generated oxygen radicals, as measured by luminol-enhanced chemiluminescence and superoxide anion production, after stimulation with the tumour, but not with untransformed, cells. The use of specific oxygen radical scavengers and inhibitors, superoxide dismutase, catalase, dimethyl sulphoxide and deferoxamine as well as the myeloperoxidase inhibitor 4-aminobenzoic acid hydrazide, indicated that chemiluminescence was dependent on the production of superoxide anion and hydroxyl radical and the presence of myeloperoxidase. The tumour cell-induced chemiluminescent response of monocytes showed different kinetics from that seen after activation of monocytes with phorbol ester. These results indicate that human monocytes can be directly stimulated by tumour cells for reactive oxygen intermediate production. Spontaneous monocyte-mediated cytotoxicity towards cancer cells was inhibited by superoxide dismutase, catalase, deferoxamine and hydrazide, implicating the role of superoxide anion, hydrogen peroxide, hydroxyl radical and hypohalite. We wish to suggest that so-called 'spontaneous' tumoricidal capacity of freshly isolated human monocytes may in fact be an inducible event associated with generation of reactive oxygen intermediates and perhaps other toxic mediators, resulting from a contact of monocytes with tumour cells.

Adenocarcinoma↗

PubMed1999

A congenital defect of the pericardium.

Ten patients with congenital defects of the pericardium were treated in Departments of Cardiac Surgery, Silesian School of Medicine in Zabrze and Katowice between 1989 and 1998. There were eight children and two adults, eight males and two females. In each case the pericardial defect was discovered intraoperatively during surgery for congenital heart defect. There were no cases with clinical symptoms that could be clearly related to the defect of the pericardial sac. In the case of a child with a complete absence of the left pericardial wall the heart was significantly rotated contrary to the defect. The final outcome of the congenital heart defect surgery was satisfactory in each case. An abbreviated historical review of the diagnosis and treatment of the pericardial defects is presented with special attention placed on therapeutic management. Surgical correction of pericardial defects is concluded to be justified in patients with clinical symptoms. In most cases pericardial defects are discovered intraoperatively, but when they are large the said defects do not require any treatment.

Adult↗

PubMed1999

Effect of the immunomodulating agent, pentoxifylline, in the treatment of sepsis in prematurely delivered infants: a placebo-controlled, double-blind trial.

OBJECTIVE: To evaluate the influence of the methylxanthine derivative, pentoxifylline, on plasma levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, and IL-6 in prematurely delivered infants with generalized bacterial infections and to assess the effect of this immunomodulating drug on the clinical outcome in newborns with sepsis. DESIGN: A prospective, randomized, double-blind trial. SETTING: The neonatal intensive therapy units in university teaching hospitals. PATIENTS: One hundred patients with sepsis admitted during a 1.5-yr period. INTERVENTIONS: Patients were randomly assigned to receive pentoxifylline (pentoxifylline group) in a dose of 5 mg/kg/hr for 6 hrs on 6 successive days or an identically presented placebo (placebo group). MEASUREMENTS AND MAIN RESULTS: Only infants with sepsis confirmed by positive blood culture were recruited into the study. There were no significant differences at randomization between the pentoxifylline and placebo groups with regard to the birth weight, gestational age, gender, Apgar score, hypotension, neutropenia, thrombocytopenia, metabolic acidosis, plasma levels of cytokines, and occurrence of shock. Plasma levels of TNF, IL-1, and IL-6 were evaluated before and after the drug or placebo administration on the first, third, and sixth days of therapy. Cytokines were determined by immunoenzymetric test EASIA (TNF) and Endogen Interleukin-Elisa (IL-1, IL-6). The frequency of gram-negative sepsis was similar in both groups (37.5% and 36.8%). Pentoxifylline significantly diminished plasma TNF levels (p = .009) but had no effect on plasma IL-1 levels. Mean plasma IL-6 levels, which were measured in the pentoxifylline group on the 6th day of the study, were significantly lower compared with respective data obtained in the placebo group. Only 1 of 40 infants with sepsis in the pentoxifylline group died, whereas 6 of 38 infants in the placebo group did not survive (p = .046). An increased incidence of disordered peripheral circulation and metabolic acidosis (p = .048), anuria or oliguria (p = .03), disseminated intravascular coagulation (p = .043), and the occurrence of clinical symptoms of necrotizing enterocolitis (p = .025) was observed in the course of sepsis in infants in the placebo group. CONCLUSION: Pentoxifylline significantly affects the synthesis of TNF and IL-6 as well as reduces the mortality rate in premature infants with sepsis. The dosage and schedule of drug administration in this study attenuated the severity of the clinical course of sepsis in this group of patients.

Adjuvants, Immunologic↗

PubMed1999

Local low-dose IL-2 therapy.

Interleukin-2 (IL-2) is a powerful drug for treating cancer. However, it is only powerful if it is properly applied. That is, IL-2 should be applied at the tumor site, because at the transition of normal and malignant tissue are the tumor infiltrating cells. These should be activated by IL-2. Local application implies that IL-2 can be used in relatively low doses. It is becoming clear that even a single injection of IL-2 can cure cancer. IL-2 can also enhance the therapeutic effects of irradiation and Cisplatin. Locally applied IL-2 therapy is virtually non-toxic.

Animals↗

PubMed1999

Preferential overexpression of CD44v5 in advanced gastric carcinoma Goseki grades I and III.

Tumor progression is associated with the clonal expansion of surviving cell variants. These results in cancer cell heterogeneity and selection of cells with a high malignant potential reflected also by the ability to metastasize. In seeding and implantation of cancer cells at the distant site cell adhesion molecules play a crucial role. Of particular interest is CD44 adhesion molecule, which possibly is involved in tumor metastasis development. Forty cases of an advanced gastric cancer were studied. Paraffin block were collected from the files. In addition to routine tumor typing, grading (Lauren and Goseki classifications) and staging, CD44 (standard, v5 and v6) was studied by immunohistochemistry and RT-PCR. CD44 immunoreactivity was found in 36 of the 40 studied cases. A significant overexpression of CD44v5 was noticed in gastric cancer. This was especially seen in Goseki's grades I and III (72.7% of cases) and was less common in Goseki's grades II and IV (44.4% of cases). CD44v6 was less commonly expressed. In some cases CD44 heterogeneity of neoplastic intravascular emboli was noticed and in some other cases stronger expression of CD44 was present in deeper parts of cancer infiltrate. Immunohistochemical expression was mostly in concert with CD44 gene expression as shown by RT-PCR results. Some discrepancies are discussed. These findings are interesting in view of better prognosis and different route of dissemination of Goseki's grades I + III compared with Goseki's grades II + IV of the gastric cancer. We have shown an overexpression of CD44v5 in an advanced gastric cancer, especially in Goseki's grades I and III. This could reflect a different malignant potential and a different route of dissemination of gastric well differentiated adenocarcinomas.

Adenocarcinoma↗

PubMed1998

Impairment of B-lymphocyte differentiation induced by dual triggering of the B-cell antigen receptor and CD40 in advanced HIV-1-disease.

OBJECTIVE: This study was performed to investigate the hyporeactivity of purified B lymphocytes from HIV-1-infected patients. DESIGN: Given the importance of the B-cell Ag receptor (BCR) and CD40 in B-lymphocyte activation, we assessed the capacity of purified peripheral blood B lymphocytes from HIV-1-infected patients to differentiate into Ig-secreting cells in a T-cell- and accessory-cell-independent system of BCR and CD40 costimulation. METHODS: B lymphocytes from 21 HIV-1-infected patients were purified by immunomagnetic cell separation and costimulated with immobilized anti-CD40 monoclonal antibodies and Staphylococcus aureus Cowan I particles in the presence of interleukin (IL)-2 and IL-10. Homotypic aggregate formation, apoptosis, cell cycle entrance, proliferation and Ig secretion of B cells were analysed. RESULTS: Costimulation by the BCR and CD40 induced proliferation and differentiation of B lymphocytes into Ig-secreting cells in 13 patients (group I) but not in eight patients (group II). For three patients in group II, the dual triggering induced apoptosis of B cells. The unexpected inability of these cells to differentiate was associated with a high CD38 expression and a weak spontaneous production of Ig or anti-HIV-1 antibodies in patients with a high viral load and a low CD4+ lymphocyte count. Despite this anomaly, the B cells from group II were able to progress through the cell cycle after stimulation with a combination of phorbol ester and ionomycin in complete medium, suggesting an impairment in BCR and CD40 early signal transduction. CONCLUSION: Intrinsic in vitro hyporeactivity of B lymphocytes to dual triggering of BCR and CD40 was observed in advanced HIV-1 disease and appeared to be related to in vivo hyperactivation of B cells.

Adult↗

PubMed1998

Tumour necrosis factor alpha (TNF alpha) and leukaemic cells: secretion and response.

Leukaemic cells from various types (ALL-O, ALL-proB, ALL-common [cALL], ALL-T) of acute lymphoblastic leukaemia (ALL) and non-lymphoblastic leukaemia (ANLL) were studied for their ability to produce tumour necrosis factor alpha (TNF). The role of exogenous and endogenous TNF in proliferation in vitro of leukaemic cells was also assessed. Leukaemic cells from different types of acute leukaemia were found to show major differences in their ability to proliferate in vitro. High production of TNF was observed mainly in ALL-T and ANLL but in ALL-T it was not associated with the proliferation rate of leukaemic cells. In most instances no bioactive TNF was detected. The variable response of leukaemic cells (inhibition or stimulation of proliferation) to exogenous human recombinant (rTNF) was assessed. The role of endogenous TNF as a growth factor or agent associated with resistance to exogenous TNF was excluded as judged by a lack of effect of the TNF synthesis inhibitor pentoxifylline (PTX) and of anti-TNF antibody and PTX/rTNF. There was no association of the effect of rTNF on proliferation of leukaemic cells with their immunophenotype, spontaneous release of TNF or proliferation rate. Hence, this study does not provide evidence for the role of TNF as a growth factor for leukaemic cells or for its role in the inhibition of proliferation of leukaemic cells.

Adolescent↗

PubMed1998

Immunohistological diagnosis of myocarditis. Potential role of sarcolemmal induction of the MHC and ICAM-1 in the detection of autoimmune mediated myocyte injury.

AIMS: The aim of this study was to characterize the up-regulation of major histocompatibility complex antigens class I (HLA-A, B, C) and class II (HLA-DR), and intercellular adhesion molecule-1 with special emphasis on de novo induction of these molecules on cardiac myocytes in patients with suspected myocarditis. METHODS: Endomyocardial biopsy specimens were obtained from 83 consecutive patients. Specimens were subdivided into two groups according to clinical presentation of the disease: Group A, 58 patients with idiopathic congestive heart failure and Group B, 25 patients without heart failure. In group A, 15% of patients had active myocarditis, 36% had borderline and 48% no myocarditis according to Dallas criteria. In group B, myocarditis distribution was 12%, 36% and 52%, respectively. The major histocompatibility complex was considered to be positive when the immunoreactivity index was > or = 3+; intercellular adhesion molecules were considered positive when the score was > or = 2+. RESULTS: We observed two characteristic staining patterns of the HLA molecules on cardiac myocytes: (i) multifocal sarcolemmal staining at the sites of mononuclear inflammatory infiltration, (ii) linear sarcolemmal staining with otherwise normal endomyocardium. Positive immunoreactivity was observed in 60% of patients in group A, and in 44% of patients in group B. We found no correlation between a histopathological diagnosis of myocarditis or left ventricular systolic function and positive immunoreactivity. CONCLUSION: The lack of correlation between immunohistological signs of active inflammation and clinical symptoms in patients with myocarditis may indicate independence of an immunological mechanism. The lack of correlation between immunoreactivity and histopathological diagnosis of myocarditis may suggest low sensitivity of traditional histological evaluation. In our opinion, the induction of major histocompatibility and intercellular adhesion molecules on cardiac myocytes may indicate an autoimmune inflammatory response in patients with inflammatory myocardial disease. Immunohistochemical methods may be helpful in selecting patients for immunosuppressive therapy.

Adult↗

PubMed1998

Intravesical interleukin-2 in T1 papillary bladder carcinoma: regression of marker lesion in 8 of 10 patients.

PURPOSE: We evaluate the therapeutic effect of intravesical interleukin-2 (IL-2) on T1 papillary bladder carcinoma after incomplete transurethral resection. MATERIALS AND METHODS: After incomplete transurethral resection we treated 10 patients in whom the marker lesion was left in place with 3 x 10(6) Chiron units IL-2 in 50 ml. saline plus 0.1% human serum albumin. The solution remained in the bladder for 2 hours and it was instilled on 5 consecutive days. The effect of IL-2 treatment on the marker lesion was evaluated by cystoscopy and repeat biopsy of the marker site 2 months after treatment. In addition, the effect on the recurrence of bladder tumors was studied. RESULTS: At 2 months 8 of the 10 marker lesions (80%) had completely regressed and there were no tumor cells on repeat biopsy. Four patients remained tumor-free after 30 to 54 months. We noted no toxic effects. In 1 patient with a 7-year history of bladder cancer the marker was only partially regressed after 2 months. After removal of the marker this patient remained tumor-free at a followup of 54 months. CONCLUSIONS: To our knowledge this report represents the first study of the effect of IL-2 on marker lesions left in place after transurethral resection. The results indicate that IL-2 instillations are feasible, and the combination of transurethral resection and IL-2 instillation may have a powerful antitumor effect. The therapeutic effects may not simply be due to intravesical IL-2, because previous transurethral resection probably caused some influx of infiltrating cells and the marker may have had tumor associated antigens. Consequently these effects may be due to the interaction of tumor associated antigens, infiltrating cells and IL-2.

Administration, Intravesical↗

PubMed1998

Inhaled corticosteroid improves bronchial reactivity and decreases symptoms in patients with mitral stenosis.

STUDY OBJECTIVE: To determine if treatment with inhaled budesonide forte can diminish increased bronchial hyperreactivity and improve symptoms in patients with mitral valve stenosis. DESIGN: The study was randomized, double blind, and placebo controlled. SETTING: Outpatient/university hospital. PATIENTS: Twelve subjects, 8 female and 4 male, who qualified for mitral valve replacement. All subjects presented with increased bronchial reactivity to histamine at the time of the study. INTERVENTIONS: Patients received placebo or budesonide forte twice a day (1,200 mg/d) for 6 weeks. During the study, patients were treated with the same doses of diuretics and other medications that could affect bronchial reactivity. MEASUREMENTS: Spirometry, provocative concentration of histamine causing a 20% fall in the FEV1 (PC20H), symptom scores. RESULTS: In the treated group, the initial PC20H was 0.82+/-0.72 mg/mL; in the placebo group 1.39+/-1.3 mg/mL. After 6 weeks of treatment, PC20H was significantly higher (3.07+/-2.28 mg/mL; p > 0.01) in the budesonide-treated group and remained unchanged in the placebo group (1.49+/-0.91). Symptom scores were significantly lower after administration of budesonide forte (mean change, 4.0+/-2.6). CONCLUSIONS: Six weeks of treatment with budesonide forte significantly decreased bronchial reactivity to histamine and improved symptoms in patients with mitral valve stenosis.

Administration, Inhalation↗

PubMed1998

Detection of cancer cells in the blood by FACS sorting of CD45- cells.

We describe a simple and sensitive method for detection of low number of cancer cells in the blood. The method is based on FACS sorting of leukocytes labelled with anti-CD45 monoclonal antibody and examining CD45- cells by conventional cytology and immunostaining for cytokeratin 18. In a model study, cancer cells seeded at the frequency of 1 per 106 and 1 per 107 leukocytes were detected in CD45- population. Sensitivity of this method was comparable to reverse transcription polymerase chain reaction (RT-PCR) used for detection of cancer cells expressing CD44 variants-mRNA. In a pilot study, cancer cells were also isolated from the blood of some patients with locally advanced gastric cancer. This method may be useful for detection of circulating tumour cells in cancer patients.

Blood Cells↗

PubMed1998

Detection of cytokine gene expression in human monocytes and lymphocytes by fluorescent in situ hybridization in cell suspension and flow cytometry.

The use of digoxigenin (DIG)- and biotin-labelled dsDNA probes to detect TNFalpha-mRNA accumulation in human peripheral blood mononuclear cells (PBMC) and isolated monocytes is described. The fragment of the glyceraldehyde-3-phosphate dehydrogenase GAPDH-cDNA was used as a control probe. The hybridization signals were detected by staining with fluorescein isothiocyanate (FITC)-labelled anti-DIG antibody and avidin-FITC, respectively. The cells were stimulated in vitro with lipopolysaccharide (LPS) for 0.5-6 h. The TNFalpha-mRNA was detected in monocytes 1 h after stimulation with LPS, and the highest accumulation was seen around 2 h. The TNFalpha-mRNA in stimulated PBMC was detected at the lower level peaking around 4 h. The TNFalpha-mRNA accumulation was lower in lymphocytes than in monocytes when PBMC were studied. There was no difference in the level of GAPDH-mRNA between unstimulated and stimulated cells. Finally, an enhanced accumulation of TNFalpha-mRNA was observed in PBMC from some patients with sepsis or cancer. Thus, this study shows that cytokine gene expression may be detected in cells ex vivo. This opens the possibility of studying the level of cytokine gene activation in PBMC of patients with diseases where the role of cytokines in their pathophysiology is implicated.

Biotin↗