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PubMed2002

Expression of interleukin-12 and its signaling molecules in peripheral blood mononuclear cells in systemic lupus erythematosus patients.

OBJECTIVE: To determine the in vitro expression of interleukin-12 (IL-12) and its effect on signal transducers and activators of transcription (STAT) signaling molecules in peripheral blood mononuclear cells (PBMCs) in patients with systemic lupus erythematosus (SLE). METHODS: Peripheral blood mononuclear cells in 39 patients with definite systemic lupus erythematosus and 11 healthy volunteers were collected. Expression of IL-12 P40mRNA in PBMCs was determined with reverse transcription-polymerase chain reaction (RT-PCR). Quantity of IL-12 protein supernatant was measured by enzyme-linked immunosorbent assay (ELISA). The levels of phosphorylated STAT3 and STAT4 signaling molecules in PBMCs were detected by immunoblot. RESULTS: Levels of IL-12 protein and mRNA expression in patients with active or inactive SLE were significantly higher than those in controls. Phytohemagglutinin (PHA ) may promote the expression of IL-12. IL-12 alone induced the phosphorylation of STAT3 and STAT4 in PBMCs from patients with SLE, especially in active SLE. However it had no obvious effect on normal PBMCs. Phosphorylated STAT3 and STAT4 might be observed in normal PBMCs treated with IL-12 plus PHA. CONCLUSION: IL-12 is produced aberrantly in patients with SLE. IL-12 might exert its biological role in SLE via the aberrantly phosphorylated STAT3 and STAT4 signaling molecules.

Adolescent↗
PubMed2003

[The influence of the LPS from Bacteroides fragilis on the secretion of IL-2 and IL-4 from the peripheral blood mononuclear cells of normal volunteers].

OBJECTIVE: To investigate the influence of the LPS of Bacteroides fragilis on the secretion of IL-2 and IL-4 from the peripheral blood mononuclear cells of normal individuals, so as to elucidate the mechanism of the infection by Bacteroides fragilis. METHODS: LPS was obtained from both the strains isolated from patients and from standard NCTC9343. Peripheral blood mononuclear cells (PBMCs) were treated with different concentrations of LPS thus obtained. The supernatants from the cell culture of the PBMCs were harvested at 24 PBHs and were subjected to the determination of the IL-2 and IL-4 contents by ELISA method. RESULTS The IL-2 secretion from the PBMCs of normal volunteers was obviously inhibited by the LPS from Bacteroides fragilis (P < 0.01), and the inhibitory effect was dose-dependent. Nevertheless, the IL-4 secretion from the PBMCs of normal volunteers was significantly stimulated by the LPS from Bacteroides Fragilis (P < 0.05), and it was not concentration dependent. There was no difference between the effects of the LPSs from patients and standard strains (P < 0.05). CONCLUSION: The LPS from Bacteroides fragilis was inhibitory to the secretion of IL-2 from PBMCs and was stimulative to that of IL-4 from PBMCs of normal human persons.

Bacteroides fragilis↗
PubMed2003

[Identification of phenotype and Interferon-alpha-producing capability of circulating type II dendritic cells and its clinical implication in HBV-infected patients].

OBJECTIVE: To investigate the number, phenotype, and interferon-alpha (INF-alpha) of type II dendritic cells (DC2) in persons with hepatitis B and evaluate the role of DC2 subset in the immunopathogenesis of chronic HBV infection. METHODS: Peripheral blood was extracted from 103 hepatitis B (HB) virus-infected persons, including 11 cases of HB virus (HBV)-infected persons, 11 cases of acute HB, 81 cases of chronic HB, and 11 cases of asymptomatic HBV infection, and 25 healthy blood donors used as controls. Flow cytometry was used to calculate the number and the phenotype of circulating DC2. Ultraviolet-inactivated herpes simplex virus (HSV)-1 was added into the suspension of peripheral blood mononuclear cells (PBMCs) and then co-cultured for 24 hours to stimulate the production of INF-alpha by DC2 that was examined by ELISA assay. RESULTS: The number of DC2 in patients with chronic HB was 3.3 +/- 1.0 10(6)/L, significantly lower than that in the healthy controls (7.2 +/- 2.4 10(6)/L, P < 0.01). However, the number of DC2 was not significantly different between any other groups. The proportion of GS2 to PBMCs in the patients with chronic HB was 1.12 +/- 1.13 approximately 0.22 +/- 0.10, all significantly lower than that in the healthy controls (0.32% +/- 0.13%, P < 0.01). However, the proportion of GS2 to PBMCs was not significantly different between any other groups. The decrease of number of DC2 and that of proportion of DC2 to PBMCs in patients with chronic HB were related with the progress of disease. The INF-alpha concentration in the suspensions of PBMCs of different groups without stimulation by HSV-1 were low and there was no significant difference in INF-alpha concentration between different groups. The INF-alpha concentration in the suspension of PBMCs of healthy controls was 789 +/- 82 pg/ml, significantly higher than those of the patients with acute HB (161 +/- 36 pg/ml) and the patients with chronic HB (183 +/- 113 pg/ml, 147 +/- 39 pg/ml, and 156 +/- 39 pg/ml, all P < 0.05). However, there was no significant difference between the patient groups (all P > 0.05). CONCLUSION: The number and INF-alpha producing function of DC2, and the numbers of NK cells and CD8+ T cells in peripheral blood of patients with chronic HB decrease significantly, which results the deficiency of HBV-specific immune response.

Adolescent↗
PubMed2003

[Inhibition of antisense-endothelin converting enzyme RNA on interleukin-5 released from dust mite-challenged peripheral blood mononuclear cells in patients with allergic asthma].

OBJECTIVE: To investigate the effect of antisense endothelin converting enzyme (ECE) RNA on levels of cytokines released from CD(4)(+) lymphocytes in patients with allergic diseases responsive to house dust mites. METHODS: Peripheral blood mononuclear cells (PBMCs) were separated from 21 patients who were sensitive to dust mites. PBMCs from those patients were divided into two groups. No stimulation group (A group) induded A(1) group (anti-ECE epithelial cells + PBMCs) and A(2) group (control cells + PBMCs). Stimulation group (B group) included B(1) group (anti-ECE epithelial cells + PBMCs + dust mites extract) and B(2) group (control cells + PBMCs + dust mites extract). House dust mite extract (20 microg/ml) was added to the culture of stimulation group as described above. After 72 hours, supernatants from both groups were collected and the levels of IL-5 and IFN-ggr; released into the supernatants were detected by enzyme-linked immunoabsorbent assay. RESULTS: IL-5 levels were increased significantly after treatment with dust mite in twelve of 21 cases. No significant differences of IL-5 were found between the groups of A(1)[(6.0 +/- 1.3) x 10(-9) g/L] and A(2) [(7.5 +/- 1.1) x 10(-9) g/L] before house dust mite stimulation in the 12 cases (P > 0.05), and no significant differences in IFN-ggr; were found between the groups of A(1) [(63 +/- 26) x 10(-9) g/L] and A(2) [(70 +/- 52) x 10(-9) g/L] before house dust mite stimulation (P > 0.05). IL-5 level was increased in both groups after stimulation but it was significantly lower in the B(1) group [(8.2 +/- 1.6) x 10(-9) g/L] than that in the B(2) [(12.0 +/- 1.8) x 10(-9) g/L] (P = 0.047). It seemed that increased IFN-ggr; level after stimulation was higher in B(2) [(153 +/- 71) x 10(-9) g/L] than that in the B(1) group (100 +/- 41) x 10(-9) g/L], but there was no statistic significance (P > 0.05). In addition, our results also showed that the release of IL-5 was significantly increased in those cases with asthma, or asthma plus allergic rhinitis after dust mites stimulation [(44 +/- 15)%] compared with that in those with urticaria [(7 +/- 4)%] (P = 0.047). CONCLUSIONS: Antisense-ECE downregulated the IL-5 secretion from Th2 lymphocytes in patients with allergic asthma after being challenged with dust mites. It is indicated that ET-1 is an important cytokine involved with allergic airway inflammation. Antisense-ECE RNA management in airways may be of value in treating allergic asthma.

Adult↗
PubMedDate not supplied

High levels of sFas and PBMC apoptosis before and after excision of malignant melanoma--case report.

In our study we investigated the level of apoptosis in PBMCs and the serological level of sFas (CD95/APO-1) in 22 patients with malignant melanoma (12 patients with unique cutaneous primary tumour and 10 patients with unique brain metastasis). The first determination was performed before tumour excision and the second at 6-7 months after excision. Results in patients with primary tumour in the first determination: 6 patients with over normal values in PBMCs apoptosis and 5 patients with increased values of sFas. In the second determination: apoptosis was increased in 5 patients and sFas level was increased in 4 cases. In patients with metastases in the first determination apoptosis of PBMC was increased in 7 cases and sFas in 5 cases. In the second determination apoptosis was increased in 4 cases and sFas was increased in 4 cases. Our results show that half of the investigated patients presented elevated values of PBMCs apoptosis and Fas receptor both before and 6-7 months after tumour excision. Apoptosis values for PBMCs and sFas values were with 1/4 higher than normals. There was no difference in clinical evolution of the patients with normal or increased values for studied parameters. Clinical evolution was performed for 1 year. The presence of increased values for PBMCs and sFas after tumour excision, primary or metastasis is surprising and hard to explain. It is possible that tumoral evolution induces a disregulation at PBMCs level or other cells level that persists unexpectedly, after tumour excision or apoptotic processes, in a certain level to be independent and anterior to tumour development.

Apoptosis↗
PubMed2004

PKR gene expression and response to pegylated interferon plus ribavirin therapy in chronic hepatitis C.

Pegylated interferon (PEG-IFN) alpha combined with ribavirin is the current standard treatment for hepatitis C, but around 50% of patients do not respond for reasons that are not fully understood. To explore the regulation of IFN-inducible protein kinase (PKR), we have measured PKR mRNA levels in peripheral blood mononuclear cells (PBMCs) and in liver biopsies from patients with chronic hepatitis C. PBMCs were also analysed after in vitro incubation with IFN and during antiviral therapy. Non-responders to PEG-IFN plus ribavirin had pre-treatment PKR mRNA levels in PBMCs (0.1+/-0.0074) and in liver (0.102+/-0.051) that were significantly higher than those of responders (PBMCs: 0.023+/-0.014, P=0.0005; liver: 0.034+/-0.020; P=0.0002). On the other hand, PKR mRNA levels in PBMCs were similar in non-responders and in responders after in vitro exposure to IFN (0.434+/-0.301 vs 0.403+/-0.222; P=NS) and during therapy (0.31+/-0.10 vs 0.30+/-0.12; P=NS). These results indicate that in hepatitis C, non-responsiveness to IFN-alpha is associated with pre-treatment up-regulation of the PKR gene, evidence that the infecting hepatitis C virus is able to stimulate endogenous IFN production, being resistant to its antiviral effect. On the other hand, the PKR gene response to exogenous IFN was similar in responders and non-responders, at least in PBMCs, suggesting that variations in its activation are not major determinants of the outcome of antiviral treatment.

Adult↗
PubMed2005

Abnormal expression of hepatomas and circulating telomerase and its clinical values.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant tumours worldwide. Telomerase is reactivated in various types of malignant tumors and may contribute to the development of HCC. To evaluate the role of telomerase in formation and development of HCC, we analyzed its expression status in different parts of HCC tissues and peripheral blood mononuclear cells (PBMCs), and explored its clinical implications for diagnosis of HCC. METHODS: Total RNAs and telomerase were extracted from HCC and their non-cancerous tissues, and both relationships were analyzed between them. The expression of telomerase reverse transcriptase (hTERT) mRNA and telomerase activities in liver tissues and PBMCs were detected by RT-PCR and telomeric repeat amplification protocol (TRAP)-ELISA, respectively. The diagnostic values of telomerase in PBMCs were investigated in the diagnosis and differentiation of HCC. RESULTS: The specific activities of telomerase were 18.25+/-15.02 A/microg RNA in HCC tissues, and significantly higher than those in their non-cancerous tissues (8.16+/-6.22 A/microg RNA, P<0.05). But total RNA levels in HCC were 12.40+/-7.34 microg/mg wet liver and lower than 53.77+/-52.02 microg/mg wet liver in their non-cancerous parts (P<0.01). The different telomerase levels could be detected in PBMCs from patients with chronic liver diseases and control group. The enzyme activities were significantly higher in HCC than in any other groups (P<0.01). However, circulating telomerase levels were more obviously decreased in HCC patients after transcatheter arterial embolization treatment than before the treatment (P<0.01). The analysis of combined serum alpha-fetoprotein level and PBMCs telomerase was more sensitive and specific for the diagnosis of HCC. CONCLUSION: The abnormal expression of telomerase in HCC tissues and circulating PBMCs could be a useful marker to the diagnosis and prognosis of HCC.

Balloon Occlusion↗
PubMed2005

[Influence of nuclear factor-kappaB activation on the expression of cytokines in monocytes stimulated by burn serum].

OBJECTIVE: To investigate the effects of NF-kappaB activation on the expression of cytokines in monocytes stimulated by burn serum, so as to explore the mechanism in monocyte activation by burn serum. METHODS: Peripheral blood monocytes (PBMCs) isolated from healthy volunteers were employed as the target cells. The cells were stimulated by serum from healthy volunteers (control), by serum from burn patients (burn serum), and by burn serum with addition of PDTC (pyrrolidine dithiocarbamate). Activation of monocytic NF-kappaB before stimulation and at 0.5, 1.0, 2.0 and 4.0 poststimulation hours (PSH) was assessed with electrophoretic mobility shift assay (EMSA). Expression of TNF-alpha and IL-8 mRNA at 1.0, 2.0, 4.0, 6.0 PSHs was assayed with in situ hybridization (ISH). Meanwhile, the contents of TNF-alpha and IL-8 in the supernatants were assayed by enzyme linked immunosorbent assay (ELISA). RESULTS: The monocytic NF-kappaB activity in burn serum group increased significantly and reached the peak level at 1 PSH [(30.2 +/- 3.5) x 10(4) integration gray scale value] after the PBMCs were stimulated by burn serum, and it was obviously higher than that in control group [(4.4 +/- 0.8) x 10(4) integration gray scale value], (P < 0.01). It gradually decreased and returned to the pre-stimulation state at 2 PSH. The monocytic NF-kappaB activity in PDTC group decreased to [(6.8 +/- 0.9) x 10(4) integration gray scale value at 1 PSH] after the stimulation. The expression of TNF-alpha mRNA of the monocytes and the TNF-alpha level in the supernatant of the cultured PBMCs reached peak level at 1 PSHs after being stimulated by burn serum, and they were obviously higher than those in control group (P < 0.01). While the expression of IL-8 mRNA and the IL-8 level in the supernatant of the cultured PBMCs reached peak level at the 4 PSHs after being stimulated by burn serum, which were obviously higher than those in control group (P < 0.01) too. In addition, the synthesis and release of TNF-alpha (peaked at 1 PSH: 0.52 +/- 0.06 microg/L) and IL-8 (peaked at 4 PSH: 239 +/- 20 ng/L) in the supernatant of PBMCs in PDTC group were evidently higher than those in control group [(0.13 +/- 0.07) microg/L, < 156 ng/L] (P < 0.01). CONCLUSION: Burn serum can induce the activation of NF-kappaB which lead to the synthesis and release of cytokines from PBMC. This result indicates that the activation of NF-kappaB plays important role in the secretion of cytokines from PBMCs induced by the burn serum.

Adolescent↗
PubMed1989

Specific binding of human alpha interferon to high-affinity cell-surface binding sites on peripheral blood mononuclear cells.

To characterize receptors for alpha interferon (IFN-alpha) on human cells, we studied the binding of radioiodinated recombinant DNA-derived human IFN-alpha to human peripheral blood mononuclear cells (PBMCs) from normal individuals and from patients with chronic type B hepatitis. At 1 degree C, binding reached equilibrium after 2 to 3 hours of incubation, and saturation of specific binding occurred at a concentration of approximately 4000 fmol/ml. Binding of labeled IFN-alpha was specific; it was inhibited by an excess of unlabeled IFN-alpha or IFN-beta but not by cholera toxin or IFN-gamma. Scatchard analysis of binding data yielded for normal PBMCs an apparent dissociation constant (Kd) of 1.54 +/- 0.49 x 10(-9) mol/L (mean +/- SD) and an apparent maximum binding capacity (Bmax) of 7.35 +/- 1.22 x 10(-11) mol/L. Corresponding values for patients with chronic type B hepatitis who had not received treatment were similar, suggesting that such patients should respond normally to endogenous interferon. Analysis of data on the binding of labeled IFN-alpha to normal PBMCs from experiments in which a high specific activity ligand or subpopulations of PBMCs had been used revealed that receptors for IFN-alpha on PBMCs are heterogenous. In patients with chronic type B hepatitis who were receiving IFN-alpha therapy, the apparent Kd was increased (3.02 +/- 0.91 x 10(-9) mol/L) without any appreciable change in the apparent Bmax or any appreciable changes in the proportions of subpopulations of PBMCs. This decreased affinity induced by IFN-alpha treatment does not necessarily reflect an effect on a single binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1985

Interleukin 2 production in B cell chronic lymphocytic leukemia.

Interleukin 2 (IL 2) production by phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) was investigated in 22 patients with active untreated B cell chronic lymphocytic leukemia (B-CLL) and in 15 healthy donors. PBMCs from healthy donors demonstrated an IL 2 synthesis of 12.4 +/- 10 U/mL. B-CLL PBMCs produced a significant amount of IL 2 (8 +/- 6.6 U/mL) despite the low percentage of T cells (13% +/- 8%) associated with this disease compared with that found in healthy donors (63% +/- 7.5%). If IL 2 production is expressed as units per milliliter per 10(4) T cells, its level in patients with B-CLL (1.1 U/mL/10(4) T cells) is five times greater than that of the controls (0.19 units). When expressed as units per milliliter per liter of blood, the B-CLL patients produce approximately 12 times as much IL 2 as controls. IL 2 production in normal controls was doubled after irradiation of PBMCs or addition of indomethacin. This increase was not seen with B-CLL PBMCs suggesting that the latter have been devoid of prostaglandin-producing normal IL 2 suppressor cells. By mixing normal or B-CLL T cells with non-T cells we found that T cells from patients with B-CLL stimulated by normal accessory cells produced the same amount of IL 2 as normal T cells. Moreover, B-CLL non-T cells (mainly B leukemic cells) produced no IL 2 themselves but played a much more efficient role in IL 2 production than did non-T cells from healthy donors. This was not due to detectable IL 1 production by these cells. The IL 2 produced by B-CLL PBMCs was partially purified and recovered in a 16,000 mol wt fraction, the same mol wt as IL 2 from normal cells.

Aged↗
PubMed1994

HIV-specific T-helper activity in seronegative health care workers exposed to contaminated blood.

OBJECTIVE: To evaluate human immunodeficiency virus (HIV) type 1-specific cellular immune responses in HIV-seronegative health care workers with occupational high-risk exposures to HIV-infected (HIV-positive) patients. DESIGN: Peripheral blood mononuclear cells (PBMCs) were obtained after occupational exposures to HIV, and PBMCs from health care workers exposed to HIV-negative patients served as controls. The PBMCs were stimulated in vitro with HIV envelope synthetic peptides. Interleukin 2 (IL-2) production was measured in a bioassay. The HIV antibody status was determined by standard enzyme-linked immunosorbent assays. Exposed individuals were also evaluated for HIV proviral DNA by polymerase chain reaction techniques. PARTICIPANTS: The PBMCs from eight health care workers with high-risk exposures and nine control health care workers were studied. RESULTS: The PBMCs from all individuals showed strong IL-2 production to control antigens, indicating intact T-helper function. Interleukin 2 production to HIV peptides was detected in PBMCs from six of eight HIV-exposed individuals, but in only one of the nine health care workers exposed to HIV-negative body fluids (P < .008). None of the HIV-exposed health care workers became infected as determined by negative HIV antibody and polymerase chain reaction analysis after follow-up evaluation that ranged from 8 to 64 weeks. CONCLUSION: Human immunodeficiency virus-specific T-helper activity was detected in six (75%) of eight HIV-negative health care workers with exposure to HIV-positive body fluids. Potent HIV-specific T-helper activity was detectable 4 to 8 weeks after the exposure and was lost in individuals followed up for 8 to 64 weeks. Three health care workers remained responsive at 8, 19, and 24 weeks. Exposure to HIV without evidence of subsequent infection appears to result in activation of cellular immunity without activation of antibody production.

Adult↗
PubMed1997

Peripheral blood mononuclear cells induce programmed cell death in human endothelial cells and may prevent repair: role of cytokines.

Human umbilical vein endothelial cells (HUVECs) undergo programmed cell death (apoptosis) after coculture with peripheral blood mononuclear cells (PBMCs) preactivated by ionizing radiation (IR) or by bacterial endotoxin (lipopolysaccharide [LPS]). Cell-to-cell contact-mediated apoptosis could be blocked in both cases by anti-tumor necrosis factor-alpha (anti-TNF-alpha) monoclonal antibody MAK195 and also by the antagonistic cytokine interleukin-10 (IL-10). Cell-free PBMC supernatants from both preactivation treatments were sufficient to trigger endothelial apoptosis. In contrast, MAK195 and IL-10 were found to be ineffective in this system, suggesting a TNF-alpha-independent mechanism. However, N-Acetylcystein, an antioxidant, fully abrogated programmed cell death mediated by the supernatant of IR-treated PBMCs, but not of LPS-treated PBMCs. Additionally, we found that coculture and cell-free supernatants of preactivated as well as untreated PBMCs caused cell cycle arrest in proliferating EC in G(0/1), which could be relieved by IL-10, but not by anti-TNF-alpha. Further analysis showed that transforming growth factor-beta, which was constitutively expressed in the supernatant of PBMCs, namely lymphocytes, was responsible for this. These data suggest a pathophysiologic model in which preactivated PBMCs cause EC damage and may prevent blood vessel repair by arresting the proliferation of ECs. This could contribute to the understanding of various clinical endothelial complications that occur after irradiation as well as in cases of endotoxemia or related inflammatory states.

Apoptosis↗
PubMed1998

Impaired induction of the CD28-responsive complex in granulocyte colony-stimulating factor mobilized CD4 T cells.

Use of the CD28/B7 costimulatory signal for T-cell activation was analyzed in granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood mononuclear cells (G-PBMCs) and in peripheral blood mononuclear cells obtained before administration of G-CSF (preG-PBMCs). CTLA4Ig inhibition of OKT3-stimulated proliferation was significantly lower in G-PBMCs compared with preG-PBMCs (39.9% +/- 5.6% and 72.2% +/- 5.4%, respectively; P < .001). Furthermore, as shown in electrophoretic mobility-shift assays, the inducible level of the T-cell transcription factor CD28 responsive complex (CD28RC) was suppressed in CD4 cells derived from G-PBMC. However, depletion of CD14 cells from G-PBMCs restored CD28RC induction to normal levels. Taken together, these findings suggest that the large number of CD14 monocytes in G-PBMCs may limit T-cell responsiveness by suppressing the induction of the CD28RC.

Abatacept↗
PubMed1998

Effect of interleukin-4 on pro-inflammatory cytokine production and the acute phase response in healthy individuals and in patients with cancer or multiple organ failure.

1. This study investigates whether previously documented effects of interleukin-4 in down-regulating pro-inflammatory cytokine production by peripheral blood mononuclear cells (PBMCs) from healthy individuals would be reproducible in PBMCs isolated from patients with multiple organ failure (acute disease model) and gastrointestinal cancer (chronic disease model). The effects of interleukin-4 on the ability of PBMC supernatants to elicit an acute phase protein response from isolated human hepatocytes were also studied. 2. Incubation of PBMCs with interleukin-4 significantly reduced both spontaneous and lipopolysaccharide-induced production of tumour necrosis factor and lipopolysaccharide-induced interleukin-6 production, demonstrating that the PBMCs from patients with acute and chronic disease are not refractory to the effects of interleukin-4. The effects of interleukin-4 on the ability of PBMCs from the groups studied to elicit an acute phase response were complex and varied both between patient groups and individual acute phase proteins. Overall, interleukin-4 reduced the potential of PBMCs to stimulate production of the positive acute phase proteins C-reactive protein, alpha1-antichymotrypsin and alpha1-acid glycoprotein.3. This work emphasizes the pleiotropic nature of cytokines and the complex regulatory mechanisms which exist. The study illustrates the difficulties in devising in vivo intervention strategies using cytokines such as interleukin-4.

Acute-Phase Reaction↗
PubMed1997

Induction of antitumor cytotoxic T lymphocytes from the peripheral blood mononuclear cells of cancer patients using HLA-A2-restricted MAGE-3 peptide in vitro.

As the basis for the application of MAGE antigens to therapeutic use, the induction of peptide-specific CTLs has been investigated by the stimulation of peripheral blood mononuclear cells (PBMCs) with antigenic peptides derived from MAGE genes. However, the cross-reactivity of the peptide-induced CTLs to the target cells endogenously presenting the MAGE epitope, especially in cancer patients, remains controversial, despite the use of complicated manipulations. Because we recently developed a new simplified method to induce peptide-specific CTLs that killed MAGE expressing tumor cells from the PBMCs of a healthy donor, we examined the induction of specific CTLs by stimulation of PBMCs with HLA-A2-restricted MAGE-3 peptide in HLA-A2(+) cancer patients whose tumors expressed MAGE-3 by using the simple method. The CTL responses could thus be induced from unseparated PBMCs by stimulation with freshly isolated, peptide-pulsed PBMCs as antigen-presenting cells and by using interleukin 7 and keyhole limpet hemocyanin for the primary culture. All CTLs induced from the PBMCs of four cancer patients tested could thus lyse the HLA-A2 target cells pulsed with the peptide, and moreover, two of the CTLs were also able to kill HLA-A2 tumor cells expressing MAGE-3 in a HLA class I and A2-restricted manner. Therefore, these findings seem to indicate that HLA-A2-restricted MAGE-3 peptide may be potentially useful for specific immunotherapy in cancer patients.

Antigens, Neoplasm↗
PubMed1998

Dendritic cell-based vaccines in the setting of peripheral blood stem cell transplantation: CD34+ cell-depleted mobilized peripheral blood can serve as a source of potent dendritic cells.

We are investigating the use of tumor-pulsed dendritic cell (DC)-based vaccines in the treatment of patients with advanced cancer. In the current study, we evaluated the feasibility of obtaining both CD34+ hematopoietic stem/ progenitor cells (HSCs) and functional DCs from the same leukapheresis collection in adequate numbers for both peripheral blood stem cell transplantation (PBSCT) and immunization purposes, respectively. Leukapheresis collections of mobilized peripheral blood mononuclear cells (PBMCs) were obtained from normal donors receiving granulocyte colony-stimulating factor (G-CSF) (for allogeneic PBSCT) and from intermediate grade non-Hodgkin's lymphoma or multiple myeloma patients receiving cyclophosphamide plus G-CSF (for autologous PBSCT). High enrichment of CD34+ HSCs was obtained using an immunomagnetic bead cell separation device. After separation, the negative fraction of mobilized PBMCs from normal donors and cancer patients contained undetectable levels of CD34+ HSCs by flow cytometry. This fraction of cells was then subjected to plastic adherence, and the adherent cells were cultured for 7 days in GM-CSF (100 ng/ml) and interleukin 4 (50 ng/ml) followed by an additional 7 days in GM-CSF, interleukin 4, and tumor necrosis factor alpha (10 ng/ml) to generate DCs. Harvested DCs represented yields of 4.1+/-1.4 and 5.8+/-5.4% of the initial cells plated from the CD34+ cell-depleted mobilized PBMCs of normal donors and cancer patients, respectively, and displayed a high level expression of CD80, CD86, HLA-DR, and CD11c but not CD14. This phenotypic profile was similar to that of DCs derived from non-CD34+ cell-depleted mobilized PBMCs. DCs generated from CD34+ cell-depleted mobilized PBMCs elicited potent antitetanus as well as primary allogeneic T-cell proliferative responses in vitro, which were equivalent to DCs derived from non-CD34+ cell-depleted mobilized PBMCs. Collectively, these results demonstrate the feasibility of obtaining both DCs and CD34+ HSCs from the same leukapheresis collection from G-CSF-primed normal donors and cancer patients in sufficient numbers for the purpose of combined PBSCT and immunization strategies.

Antigens, CD34↗
PubMed1998

Human herpesvirus 8 in Italian HIV-seronegative patients with Kaposi sarcoma.

OBJECTIVE: To evaluate the prevalence of human herpesvirus 8 (HHV-8) DNA detection in a large series of human immunodeficiency virus-seronegative patients with and without Kaposi sarcoma (KS) from the central and southern regions of Italy where classic KS is prevalent. DESIGN: Samples of lesional, peripheral unaffected, and distant normal skin and peripheral blood mononuclear cells (PBMCs) from 33 patients with KS and PBMCs from 42 control subjects were analyzed using single and nested polymerase chain reaction techniques for the presence of HHV-8 DNA. PATIENTS: A total of 33 patients with KS not related to acquired immunodeficiency syndrome (26 patients with classic KS and 7 patients with iatrogenic KS) were studied. Furthermore, 2 control groups were enrolled. The first group consisted of 13 healthy volunteers, the second of 29 patients affected by different dermatological diseases. RESULTS: Human herpesvirus 8 sequences were found in 100% of lesional and perilesional specimens, in 33% of the distant normal skin samples, and in 69.6% of the PBMCs from patients with KS. A possible correlation between HHV-8 DNA in PBMCs and the clinical stage of the disease was observed. Moreover, the prevalence of viral DNA in PBMCs from the total control group was 23.8%. No viral DNA was detected in tissue biopsy specimens taken from the control group. CONCLUSIONS: Our data suggest that HHV-8 could be a widespread virus, at least in Mediterranean regions where KS is more prevalent, such as southern and central Italy. As with other herpesviruses, it may be present lifelong in latent form somewhere in the body and may contribute to the pathogenesis of KS when other predisposing conditions are present.

Aged↗
PubMed1997

Comprehensive investigation of the presence of JC virus in AIDS patients with and without progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML), a viral-induced demyelinating disease, is becoming relatively common, while many diagnostic and pathogenetic aspects remain to be clarified. A study was therefore undertaken in 64 AIDS patients suffering from various neurological disorders, including PML (12 subjects), with the specific objective of searching for JC virus (JCV) DNA by nested PCR (n-PCR) in cerebrospinal fluid (CSF), peripheral blood mononuclear cells (PBMCs), and urine collected from all patients. CSF examination, CD4 and CD8 counts, neurological examinations, and neuroradiological investigations were undertaken. JCV DNA was detected in 92% of CSF specimens in 75% of the PBMCs and urine samples from the PML patients, whereas among the non-PML patients JCV DNA was not detected in any CSF samples, but was found in 10% of PBMCs and in 39% of the urine specimens. BKV and JCV DNA viruria was observed simultaneously in 6% of the AIDS patients without PML. The routine CSF tests including IgG oligoclonal bands, the Link, and Tourtellotte IgG indexes, did not show a typical pattern in PML cases. The data obtained clearly indicate that the detection of JCV DNA in CSF constitutes an efficient marker for PML diagnosis. The simultaneous presence of JCV DNA in the CSF, PBMCs, and urine samples from the PML patients, who did not differ from controls with regard to their immunosuppressive status, suggests that JCV could be carried into the central nervous system (CNS) by infected PBMCs.

AIDS-Related Opportunistic Infections↗