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PubMed1998

Endotoxin binding and elimination by monocytes: secretion of soluble CD14 represents an inducible mechanism counteracting reduced expression of membrane CD14 in patients with sepsis and in a patient with paroxysmal nocturnal hemoglobinuria.

Little is known about the role of peripheral blood mononuclear cells (PBMCs) in lipopolysaccharide (LPS) elimination. We studied the endotoxin elimination capacities (EEC) of PBMCs of 15 healthy volunteers, 13 patients with sepsis, and 1 patient suffering from paroxysmal nocturnal hemoglobinuria (PNH). Although expression of CD14, the best-characterized receptor for LPS to date, was reduced from 93.6% +/- 0.8% in healthy subjects to 50.5% +/- 6.5% in patients with sepsis and was 0.3% in a patient with septic PNH, EEC were found to be unchanged. There was no difference in the amount of tumor necrosis factor alpha (TNF-alpha) released by PBMCs of healthy donors and patients with sepsis. Anti-CD14 antibodies (MEM-18) completely suppressed EEC, binding of fluorescein isothiocyanate-labeled LPS to monocytes as determined by FACScan analysis, and TNF-alpha release in all three groups studied. The concentrations of soluble CD14 (sCD14) secreted by endotoxin-stimulated PBMCs from healthy donors and patients with sepsis amounted to 4.5 +/- 0.4 and 20.1 +/- 1.8 ng/ml, respectively. Based on our results, we suggest that PBMCs eliminate LPS by at least two different mechanisms; in healthy subjects, the membrane CD14 (mCD14) receptor is the most important factor for LPS elimination, while in patients with sepsis (including the septic state of PNH), increased sCD14 participates in LPS elimination. Secretion of sCD14 is strongly enhanced under conditions of low expression of mCD14 in order to counteract the reduction of mCD14 and maintain the function of monocytes. This sCD14 may substitute the role of mCD14 in LPS elimination during sepsis. The elimination of LPS by PBMCs correlates with the binding reaction and the secretion of TNF-alpha.

Adult↗
PubMed1997

Accumulation of defective viral genomes in peripheral blood mononuclear cells of human immunodeficiency virus type 1-infected individuals.

Human immunodeficiency virus type 1 (HIV-1) genomes present in peripheral blood mononuclear cells (PBMCs) of infected persons or in lymphocytes infected in vitro were studied by long-distance PCR (LD-PCR) using primers localized in the HIV-1 long terminal repeats. The full-length 9-kb DNA was the only LD-PCR product obtained in peripheral and cord blood lymphocytes from seronegative donors infected in vitro. However, a high proportion (27% to 66%) of distinct populations of extensively deleted HIV-1 genomes of variable size was detected in PBMCs of 15 of 16 HIV-1-infected persons. Physical mapping of defective genomes showed that the frequency of deletions is proportional to their proximity to the central part of HIV-1 genome, which is consistent with a deletion mechanism involving a single polymerase jump during reverse transcription. Sequencing of deletion junctions revealed the presence of short direct repeats of three or four nucleotides. The number of defective HIV-1 genomes decreased after in vitro activation of PBMCs. Persistence of full-length and deleted genomes in in vitro activated PBMCs correlated with isolation of an infectious virus. Our results represent the first quantitative assessment of intragenomic rearrangements in HIV-1 genomes in PBMCs of infected persons and demonstrate that, in contrast to in vitro infection, defective genomes accumulate in PBMCs of infected persons.

Adult↗
PubMed1999

Regulation of bovine leukemia virus tax and pol mRNA levels by interleukin-2 and -10.

Recently, particular cytokines have been identified to affect progression of a variety of diseases and retrovirus infections. Previously, we demonstrated that interleukin-2 (IL-2), IL-12, and gamma interferon increased in peripheral blood mononuclear cells (PBMCs) from animals with early disease and decreased in PBMCs from animals with late disease stages of bovine leukemia virus (BLV) infection. In contrast, IL-10 increased with disease progression. To examine the effects of these cytokines on BLV expression, BLV tax and pol mRNA and p24 protein were quantified by competitive PCR and immunoblotting, respectively. IL-10 inhibited BLV tax and pol mRNA levels in BLV-infected PBMCs; however, the inhibitory effect of IL-10 was prevented in PBMCs depleted of monocytes and/or macrophages (monocyte/macrophages). To determine whether these factors were secreted or monocyte/macrophage associated, monocyte/macrophage-depleted PBMCs were cultured with isolated monocyte/macrophages in transwells where contact between monocyte/macrophages and nonadherent PBMCs was blocked. BLV tax and pol mRNA levels increased in transwell cultures similar to cultures containing nonseparated cells, and IL-10 addition inhibited the increase of BLV tax and pol mRNA. These results suggest that monocyte/macrophages secrete soluble factor(s) that increases BLV mRNA levels and that secretion of these soluble factor(s) could be inhibited by IL-10. In contrast, IL-2 increased BLV tax and pol mRNA and p24 protein production. Thus, IL-10 production by BLV-infected animals with late stage disease may serve to control BLV mRNA levels, while IL-2 may increase BLV mRNA in the early disease stage. To determine a correlation between cell proliferation and BLV expression, the effect of IL-2 and IL-10 on PBMC proliferation was tested. As anticipated, IL-2 stimulated while IL-10 suppressed antigen-specific PBMC proliferation. The present study, combined with our previous findings, suggests that increased IL-10 production in late disease stages suppresses BLV mRNA levels, while IL-2-activated immune responses stimulate BLV expression by BLV-infected B cells.

Animals↗
PubMedDate not supplied

IL-10 induces a Th2 cell tolerance in allergic asthma.

BACKGROUND: Interleukin (IL)-10 induces a long-term antigen-specific anergy in human CD4+ T cells. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from house dust mite (Dermatophagoides farinae, Der f)-sensitized asthmatic patients. PBMCs were stimulated with Der f antigen for 72 h immediately after purification or after 48 h of resting culture with medium, and IL-10 and IL-5 in the culture supernatant were measured. PBMCs were also stimulated with Der f antigen for 7 days either immediately after purification or after 48 h of resting culture, after which cells were collected. Secondary proliferative responses of these cells to stimulation for 3 days with Der f antigen and mitomycin C-treated PBMCs as antigen-presenting cells or with phorbol myristate acetate (PMA) plus calcium ionophore were investigated. RESULTS: Stimulation of PBMCs with Der f antigen immediately after purification significantly increased the proliferative response and IL-5 production. Stimulation of PBMCs with Der f antigen after resting culture with medium alone for 48 h significantly decreased IL-5 production and markedly increased IL-10 production. Although activation of cells with Der f antigen immediately after purification significantly increased secondary proliferative responses, stimulation after 48 h of resting culture failed to increase secondary proliferative responses. However, proliferation recovered when cells were activated with PMA plus calcium ionophore. CONCLUSION: These results suggest that antigen-specific Th2 cells are anergized by IL-10 and that Th2 cell tolerance may suppress eosinophilic inflammation in allergic asthma.

Allergens↗
PubMed2003

Differences in release and determination of subtype of porcine endogenous retroviruses produced by stimulated normal pig blood cells.

OBJECTIVE: Porcine endogenous retroviruses (PERVs) are of particular concern with xenotransplantations using pig cells, tissues or organs as they are present in the genome of all pig strains and are able to infect human cells in vitro. However, it remains unclear whether PERV particles will be produced in vivo and whether they may infect xenotransplant recipients. Since normal pig peripheral blood mononuclear cells (PBMCs) may be transmitted together with the transplanted organ, the production of PERVs by stimulated PBMCs was studied in vitro. METHODS: To simulate antigen-induced activation of PBMCs, phytohaemagglutinin (PHA), a T cell mitogen, and the phorbol ester O-tetradecanoylphorbol-13-acetate (TPA), a tumour promoter, were used. Virus release was estimated by measuring reverse transcriptase (RT) activity and by RT-PCR of pelleted viruses. RESULTS: Treatment of pig PBMCs with PHA or TPA induced the release of PERVs. For the first time, a correlation between the extent of proliferation of pig PBMCs and PERV production was shown. In addition, PERV release by non-proliferating cells and differences in virus production between stimuli as well as between different pig strains and individuals of one strain were observed. Subtype analysis revealed the release of the three subtypes PERV-A, PERV-B and PERV-C. In contrast to murine endogenous retroviruses, PERVs were induced by PHA alone. CONCLUSION: The data suggest that the PBMCs transmitted within a xenotransplant may release PERV. These data also suggest that pig strains producing low amounts of virus could be more suitable for xenotransplantation.

Animals↗
PubMedDate not supplied

Immunoregulation by parenteral lipids: impact of the n-3 to n-6 fatty acid ratio.

BACKGROUND: The immune system is reported to be influenced by polyunsaturated fatty acids. Therefore, immunoregulation caused by intravenous fat emulsions with different n-3 to n-6 fatty acid ratios was studied in an in vivo model. METHODS: Experimental rat heart allotransplantation served as a defined immunologic challenge. Twenty percent emulsions of safflower oil (n-3 to n-6 = 1:370), fish oil (n-3 to n-6 = 7.6:1), and soybean oil (n-3 to n-6 = 1:6.5), and a 1:1 mixture of safflower oil and fish oil (n-3 to n-6 = 1:2.1) were continuously infused (9 g of fat per kg of body weight per day) after transplantation until complete rejection. The prolongation of graft survival, an accepted parameter of immunosuppression, was assessed. Beyond that, cytokine release by mitogen-stimulated peripheral-blood mononuclear cells (PBMCs) from animals exsanguinated on day 4 after transplantation was evaluated. RESULTS: The mean rejection time was 7.8 days in the sham-infused saline control group and 6.7 days in the safflower- and fish-oil-mixture group (oil control group). Continuous infusion of soybean oil prolonged the graft survival time to 10.4 days, fish oil to 12.3 days, and safflower oil to 13.3 days. PBMC alpha-tumor necrosis factor release was significantly reduced in the fish-oil group (51.9 +/- 13.0 pg/10(6) PBMCs vs 70.8 +/- 10.9 pg/10(6) PBMCs [controls], p < .004). Interleukin-6 release was diminished in both the fish-oil group (22.2 +/- 13.6 pg/10(6) PBMCs vs 40.7 +/- 8.3 pg/10(6) PBMCs [controls], p < .002) and the safflower-oil group (28.4 +/- 6.9 pg/10(6) PBMCs, p < .002). CONCLUSIONS: The n-3 to n-6 fatty acid ratio determined the immunoregulatory potential of intravenous fat emulsions in vivo. Both n-3 and n-6 fatty acids were immunosuppressive when applied as the main polyunsaturated fatty acid sources. PBMC cytokine release was significantly reduced in these groups. The more balanced the n-3 to n-6 ratios, the less immunosuppressive the fat emulsion. There was no immunosuppressive effect at an n-3 to n-6 ratio of 1:2.1.

Animals↗
PubMed2004

(Pre-)osteoclasts induce retraction of osteoblasts before their fusion to osteoclasts.

UNLABELLED: Precursors of osteoclasts seeded on top of a confluent layer of osteoblasts/bone lining cells induced retraction of the latter cells. The (pre)osteoclasts then migrated in the formed cell-free areas and fused to form osteoclast-like cells. Retraction of the osteoblasts/bone lining cells proved to depend on activity of matrix metalloproteinases, and TGF-beta1 prevented the retraction. INTRODUCTION: It is well known that osteoblasts have a profound effect on (pre)osteoclasts in inducing the formation of bone-resorbing osteoclasts. Whether, on the other hand, (pre)osteoclasts also modulate osteoblast activity is largely unknown. Because osteoblasts/bone lining cells have to retract from the surface before resorption of bone by osteoclasts, we addressed the question of whether (pre)osteoclasts have the capacity to induce such an activity. MATERIALS AND METHODS: Rabbit calvarial osteoblasts/bone lining cells or periosteal fibroblasts were cultured until confluency, after which rabbit peripheral blood mononuclear cells (PBMCs) were seeded on top of them. The co-cultures were maintained for up to 15 days in the presence or absence of the cytokines transforming growth factor (TGF)-beta1 and TNF-alpha and selective inhibitors of matrix metalloproteinases and serine proteinases. The formation of cell-free areas and the number of TRACP+ multinucleated osteoclast-like cells were analyzed. In addition, formation of cell-free areas was analyzed in co-cultures of osteoblasts with mature osteoclasts. RESULTS: The seeding of PBMCs on a confluent layer of osteoblasts/bone lining cells resulted in the following sequence of events. (1) A low number of PBMCs strongly attached to osteoblasts. 2) At these sites of contact, the osteoblasts retracted, thus forming cell-free areas. (3) The PBMCs invaded these areas and attached to the surface of the well, after which they fused and formed multinucleated TRACP+ osteoclast-like cells. Retraction was only seen if the cells were in direct contact; conditioned media from cultured PBMCs added to osteoblasts had no effect. Mature osteoclasts seeded on osteoblasts similarly induced retraction, but this retraction occurred at a much faster rate (within 2 days) than the retraction effectuated by the osteoclast precursors (after 8 days in co-culture). Inhibition of matrix metalloproteinase activity, but not of serine proteinases, strongly reduced retraction of the osteoblasts, thus indicating that this type of cell movement depends on the activity of matrix metalloproteinases. A similar inhibitory effect was found with TGF-beta1. TNF-alpha had no effect on osteoblast retraction but enhanced the formation of multinucleated osteoclast-like cells. Addition of PBMCs to confluent layers of periosteal fibroblasts resulted in similar phenomena as observed in co-cultures with osteoblasts. However, the cell-free areas proved to be significantly smaller, and the number of multinucleated cells formed within cell-free areas was three to four times lower. CONCLUSION: Our results indicate that osteoclast precursors and mature osteoclasts have the capacity to modulate the activity of osteoblasts and that, yet unknown, membrane-bound signaling molecules are essential in inducing retraction of osteoblasts and the subsequent formation of cell-free areas.

Acid Phosphatase↗
PubMed2006

Cytokines and vascular permeability: an in vitro study on human endothelial cells in relation to tumor necrosis factor-alpha-primed peripheral blood mononuclear cells.

Tumor response is strongly enhanced by addition of tumor necrosis factor (TNF)-alpha to chemotherapy in local-regional perfusion. TNF primarily targets the endothelial lining of the tumor-associated vasculature, thereby improving permeability of the vascular bed. This augments uptake of the coadministered chemotherapeutic drug in the tumor. In vitro, however the high dose of TNF did not directly affect endothelial cells, indicating that other factors, most likely TNF-induced, are involved in the antivascular activities observed in vivo. This is supported by in vivo studies in our laboratory in which depletion of leukocytes resulted in loss of the antivascular activity of TNF. The present study examined the role of peripheral blood mononuclear cells (PBMCs) on endothelial cells by exposing them to TNF, interferon (IFN)-gamma, and PBMCs. We observed morphological changes of the endothelial cells when exposed to TNF in combination with IFN. Endothelial cells became elongated. and gaps between the cells formed. Addition of PBMCs enhanced these alterations. The endothelial layer became disrupted with highly irregular-shaped cells displaying large gap formations. PBMCs also contributed to an increased permeability of the endothelial layer without augmenting apoptosis. Replacing PBMC by interleukin (IL)-1beta produced similar effect with regard to inhibition of cell growth, morphological changes, and induction of apoptosis. Blocking IL-1beta with a neutralizing antibody diminished the effects inflicted of PBMCs. These observations indicate that endogenously produced IL-1beta by primed PBMCs plays an important role in the antivascular effect of TNF.

Apoptosis↗
PubMed1997

Fusobacterium nucleatum T18 aggregates human mononuclear cells and inhibits their PHA-stimulated proliferation.

In previous studies Fusobacterium nucleatum has been shown to induce either stimulatory or inhibitory effects on human mononuclear cells. We examined the interaction of human mononuclear cells with human and cynomolgus monkey strains of F. nucleatum. Peripheral blood mononuclear cells (PBMCs) isolated from normal donors were aggregated in the presence of cells of F. nucleatum but not control bacteria. The aggregation of PBMCs and F. nucleatum T18 was inhibited by either L-arginine, L-lysine, or pretreatment of the bacterial cells with heat, but was unaffected by the presence of sugars or normal human serum. Strain T18 aggregated purified T-cells and monocytes at approximately equal concentrations. When F. nucleatum T18 was incubated with PHA-stimulated PBMCs, DNA synthesis in the PBMCs was significantly inhibited and detection of IL-2R alpha on the PBMCs was reduced. These studies indicate that F. nucleatum aggregates PBMCs, and that this interaction is associated with both an inhibition of PBMC proliferation and a decrease in IL-2 receptor expression. The ability of F. nucleatum to inhibit mononuclear cell proliferation may be significant in the pathogenesis of periodontal diseases.

Animals↗
PubMed2000

Chlamydia pneumoniae inhibits apoptosis in human peripheral blood mononuclear cells through induction of IL-10.

Chlamydia pneumoniae is a common cause of pulmonary infection, with serum positivity in at least 50% of the general population. In this study, we report that human PBMCs exposed to C. pneumoniae are resistant to apoptosis induced by the potent photoactivated chemotherapeutic agents 8-methoxypsoralen and hypericin. In contrast, PBMCs treated with a heat-inactivated inoculum exhibit normal susceptibility to apoptosis. We also observed that human PBMCs are responsive to C. pneumoniae infection by secretion of key immune regulatory cytokines, including IL-12 and IL-10. While IL-12 may play an important role in limiting C. pneumoniae proliferation within cells, IL-10 serves an anti-inflammatory function by down-regulating proinflammatory cytokines such as IL-12 and TNF-alpha. Depletion of endogenous IL-10, but not of IL-12, abolished the apoptosis resistance of C. pneumoniae-infected PBMCs. Furthermore, addition of exogenous IL-10, but not IL-12, significantly increased the resistance of control inoculum-treated PBMCs to photoactivated 8-methoxypsoralen- and hypericin-induced apoptosis. Therefore, we conclude that C. pneumoniae possesses an antiapoptotic mechanism. The resistance to apoptosis observed in PBMCs exposed to C. pneumoniae is due, at least partially, to the IL-10 induced during C. pneumoniae infection.

Anthracenes↗
PubMed2001

High-dose leptin activates human leukocytes via receptor expression on monocytes.

Leptin is capable of modulating the immune response. Proinflammatory cytokines induce leptin production, and we now demonstrate that leptin can directly activate the inflammatory response. RNA expression for the leptin receptor (Ob-R) was detectable in human PBMCs. Ob-R expression was examined at the protein level by whole blood flow cytometry using an anti-human Ob-R mAb 9F8. The percentage of cells expressing leptin receptor was 25 +/- 5% for monocytes, 12 +/- 4% for neutrophils, and 5 +/- 1% for lymphocytes (only B lymphocytes). Incubation of resting PBMCs with leptin induced rapid expression of TNF-alpha and IL-6 mRNA and a dose-dependent production of TNF-alpha and IL-6 by monocytes. Incubation of resting PBMCs with high-dose leptin (250 ng/ml, 3-5 days) induced proliferation of resting cultured PBMCs and their secretion of TNF-alpha (5-fold), IL-6 (19-fold), and IFN-gamma (2.5-fold), but had no effect on IL-4 secretion. The effect of leptin was distinct from, and additive to, that seen after exposure to endotoxin or activation by the mixed lymphocyte reaction. In conclusion, Ob-R is expressed on human circulating leukocytes, predominantly on monocytes. At high doses, leptin induces proinflammatory cytokine production by resting human PBMCs and augments the release of these cytokines from activated PBMCs in a pattern compatible with the induction of Th1 cytokines. These results demonstrate that leptin has a direct effect on the generation of an inflammatory response. This is of relevance when considering leptin therapy and may partly explain the relationship among leptin, proinflammatory cytokines, insulin resistance, and obesity.

Adult↗
PubMed1999

Spontaneous ex vivo apoptosis of peripheral blood mononuclear cells in patients with head and neck cancer.

Proportions of apoptotic (TUNEL+) peripheral blood mononuclear cells (PBMCs) were measured by flow cytometry in patients with head and neck cancer and normal controls at the time of blood draws (0 time) and after 24-h incubation. PBMCs were incubated at 37 degrees C in medium (spontaneous apoptosis) and in the presence of CH-11 antibody (anti-Fas) or tumor necrosis factor (TNF)-alpha, both capable of inducing DNA fragmentation in activated T cells expressing the TNF family of receptors. PBMCs obtained from the patients had significantly higher (P < 0.0001) proportion of apoptotic cells than PBMCs of controls at 0 time as well as after 24-h incubation. Ex vivo apoptosis included all subsets of PBMCs: CD3+ T cells, CD16+ CD56+ natural killer cells, CD19+ B cells, and CD14+ monocytes, as determined by two-color flow cytometry. However, T cells represented the largest PBMC subset undergoing apoptosis, and lymphocytes rather than monocytes were the major TUNEL+ PBMC population. Among T cells, the level of spontaneous ex vivo apoptosis was nearly as high as that of CH-11 antibody-induced or TNF-alpha-induced apoptosis, indicating that activated Fas+ and TNFR1+ T cells were preprogrammed in vivo to die. Also, elevated levels of spontaneous apoptosis at time 0 in patients with head and neck cancer (P < 0.0001) indicated that a higher fraction of PBMCs was undergoing apoptosis in vivo in patients than controls. Together, the data suggest that an increased rate of turnover of lymphocytes is associated with cancer and may be responsible for functional lymphocyte imbalance, even in treated patients who have no evident disease.

Adult↗
PubMed1999

Is hydroxylamine-induced cytotoxicity a valid marker for hypersensitivity reactions to sulfamethoxazole in human immunodeficiency virus-infected individuals?

Hypersensitivity (HS) reactions to sulfonamides and sulfones continue to limit their use in human immunodeficiency virus (HIV)-infected individuals. In vitro cytotoxicity of hydroxylamine metabolites toward peripheral blood mononuclear cells (PBMCs) has been proposed as a marker for these HS reactions. To test the validity of this in vitro system, we determined the selective susceptibility of PBMCs from HIV-infected patients to the cytotoxic effects of hydroxylamine metabolites of sulfamethoxazole (SMX) and dapsone (DDS). Concentration-cytotoxic response data were collected using PBMCs from 12 sulfa-HS (10 SMX-HS and 2 SMX/DDS-HS) and 10 sulfa-tolerant HIV-infected individuals. Although sulfamethoxazole hydroxylamine (SMX-NOH) and dapsone hydroxylamine (DDS-NOH) both caused concentration-dependent increases in cell death, DDS-NOH was significantly more potent in each subject (P <.0001). A comparison of a variety of mean data for sulfa-HS and -tolerant patient populations failed to demonstrate the increased susceptibility of PBMCs from HS patients, noted by others, to either SMX-NOH or DDS-NOH. Moreover, any trend toward an increased susceptibility of PBMCs from HS patients was eliminated when adjusted for control cell death. PBMCs from sulfa-HS patients showed significantly greater susceptibility to the stress of short term in vitro incubation (P <. 02). Mean (S.D.) vehicle control cell death values were 24.1% (7.6%) for HS patients and 17.1% (4.4%) for tolerant patients. No significant correlation was observed between hydroxylamine-induced or control cell death and any of the recorded clinical parameters. Although several potential reasons are proposed to explain the disparity with past investigations, the data suggest that in vitro cytotoxicity is not a valid marker for HS reactions in HIV-infected individuals using currently accepted experimental procedures.

Adult↗
PubMedDate not supplied

Two-dimensional analysis of production of IL-6 and TNF-alpha can predict the efficacy of IFN-alpha therapy.

BACKGROUND/AIMS: Many patients with chronic hepatitis C are now treated with interferon-alpha (IFN-alpha). The increase in the number of patients treated with IFN-alpha, however, has resulted in increased reports of adverse drug reactions (ADR). This prompted us to investigate for a useful parameter for predicting the effects of IFN-alpha treatment before initiating the therapy for the benefit of patients. METHODOLOGY: Peripheral blood mononuclear cells (PBMCs) obtained from patients with chronic hepatitis C and healthy volunteers were incubated at 37 degrees C for 24 hours at various concentrations of IFN-alpha (1, 10(2), and 10(4) IU/ml). The tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) formed in the culture medium were determined. Also, the binding of 125I-labeled IFN-alpha to PBMCs and 2', 5'-oligoadenylate synthetase (2-5AS) activities were measured. RESULTS: The addition of IFN-alpha to PBMCs at concentrations of 1-10(4) IU/ml showed dose-dependent changes in the binding of 125I-labeled IFN-alpha to PBMCs and 2-5AS activities. There was a strong correlation between the production of IL-6 and that of TNF-alpha (r>0.9). The production of IL-6 by the PBMCs of the high responders is significantly higher than that of the low-responders at the same value of TNF-alpha. A statistically significant difference was demonstrated by analysis of covariance. CONCLUSIONS: The findings of this study suggest that two-dimensional analysis of the in vitro production of TNF-alpha and IL-6 by PBMCs induced by IFN-alpha is useful for predicting the outcome of the IFN-alpha treatment in patients.

Adult↗
PubMed2000

In multiple myeloma, circulating hyperdiploid B cells have clonotypic immunoglobulin heavy chain rearrangements and may mediate spread of disease.

DNA aneuploidy characterizes a proportion of malignant bone marrow (BM)-localized plasma cells in multiple myeloma (MM). This analysis shows that for most MM patients, circulating clonotypic B cells in MM are also hyperdiploid. Although all normal B cells and some malignant B cells are diploid, hyperdiploidy is likely to be exclusive to those that are malignant. Hyperdiploid MM B cells express CD34 and have clonotypic IgH transcripts, confirming them as part of the malignant clone. For MM, 92% (70/76) of patients had a DNA hyperdiploid subset [5-30% of peripheral blood mononuclear cells (PBMCs)] of CD19+ B cells. All CD19+ PBMCs in MM expressed CD19 and IgH variable diversity joining (VDJ) transcripts, confirming them as B cells. DNA aneuploid cells were undetectable in T or B lymphocytes from normal blood, spleen or thymus, or in blood from patients with B chronic lymphocytic leukemia. In MM, untreated patients had the highest DNA index (1.12). DNA hyperdiploid PBMCs were most frequent among untreated patients and were significantly reduced after chemotherapy. Diploid B cells were significantly more frequent after chemotherapy than at diagnosis. Of the hyperdiploid PBMCs, 81 +/- 3% expressed CD34 and CD19. In contrast to circulating CD34+ B cells, CD34- B cells in MM are diploid. In MM, unlike hyperdiploid PBMC B cells, hyperdiploid BM plasma cells lack both CD34 and CD19, suggesting that loss of CD34 correlates with differentiation and BM anchoring. In situ reverse transcription-PCR of the CD34+ (hyperdiploid) and CD34- (diploid) PBMC B-cell subsets was performed using patient-specific primers to amplify clonotypic IgH VDJ transcripts. Confirming previous work, CD34+ hyperdiploid MM PBMCs were clonotypic (86 +/- 5%). In contrast, CD34- diploid MM PBMCs had few monoclonal cells (4.8 +/- 2%). The lack of hyperdiploidy, together with the relative absence of cells having clonotypic transcripts, suggests these polyclonal CD34- B cells are normal. After culture in colchicine to arrest mitosis, hyperdiploid B cells were reduced and MM B cells accumulated in a diploid G2-M, suggesting that hyperdiploid in MM may represent a transient S-phase arrest rather than an aneuploid G0 phase. The DNA hyperdiploidy of CD34+ clonotypic B cells suggests these cells may be clinically important constituents of the myeloma clone and that they may play a direct role in the spread of myeloma.

B-Lymphocytes↗
PubMed2001

Long-term-impaired expression of nuclear factor-kappa B and I kappa B alpha in peripheral blood mononuclear cells of trauma patients.

Nuclear factor (NF)-kappa B expression and dimer characteristics were studied in peripheral blood mononuclear cells (PBMCs) of major-trauma patients and healthy controls. Analysis of PBMCs on days 1, 3, 5, and 10 after trauma revealed that expression of both p65p50 heterodimers and p50p50 homodimers was significantly reduced compared with that in controls. In vitro lipopolysaccharide (LPS) stimulation of PBMCs induced NF-kappa B translocation. However, throughout the survey, p65p50 activation remained significantly lower in trauma patients than in controls. After LPS stimulation in vitro, the p65p50/p50p50 ratio was significantly lower in PBMCs from trauma patients than from healthy controls. The ex vivo expression of I kappa B alpha was higher in PBMCs of controls than of trauma patients. LPS did not induce I kappa B expression in PBMCs from trauma patients, but strong induction was obtained with staphylococci, suggesting that this defect is not universal and depends on the nature of the activating signal. Although no direct correlation was found between levels of interleukin-10 or transforming growth factor-beta and NF-kappa B, these immunosuppressive cytokines were significantly elevated in trauma patients by 10 days after admission. The long-term low-basal and LPS-induced nuclear translocation of NF-kappa B recalled long-term immunoparalysis observed in patients with severe inflammatory stress such as trauma.

Adolescent↗
PubMed2001

Differential degradation rates of inactivated alkyltransferase in blood mononuclear cells and tumors of patients after treatment with O(6)-benzylguanine.

O(6)-Alkylguanine-DNA alkyltransferase (AGT) repairs O(6)-alkylating DNA adducts generated by alkylating therapeutic agents. Therefore, AGT activity may be an important marker of tumor and normal tissue sensitivity to chemotherapeutic agents and a predictor for the success of chemotherapeutic regimens. It is rapidly inactivated by O(6)-benzylguanine (BG) that mimics its substrates, O(6)-methylguanine and O(6)-chloroethylguanine DNA adducts. In a Phase I clinical trial, BG was given in increasing doses (from 10 to 120 mg/m(2)) by 1-h infusion. We previously reported depletion of AGT activity, and in this report, we demonstrate the relationship between degradation of BG-inactivated AGT protein and the depletion of AGT activity in peripheral blood mononuclear cells (PBMCs) and tumor samples obtained by computed tomography-guided cutting needle biopsy from patients prior to BG and either 2 or 18 h after BG. In PBMCs, BG inactivated AGT activity by over 95-100% at the end of a 1-h infusion, and depletion was maintained for 18 h. In contrast, AGT protein remained almost unchanged for up to 18 h after BG, suggesting that inactivated AGT proteins remain immunoreactive and are not rapidly degraded in PBMCs. In patient tumor biopsies, AGT activity was depleted approximately 90% 2 h after BG. Tumor AGT protein levels were reduced to approximately 40% of pretreatment values when detected by either Western blot or immunohistochemistry staining. In tumor samples obtained 18 h after BG, >95% inactivation of tumor AGT activity was observed at BG doses of 36-80 mg/m(2), and complete depletion of tumor AGT activity occurred at 120 mg/m(2) BG. However, residual AGT protein (5-10% of baseline) was detectable in all tumor samples. Therefore, the degradation of BG-inactivated AGT protein appeared to be much more rapid in tumors than that in PBMCs, which may impact on AGT regeneration rates as well. Because degradation of BG-inactivated AGT takes place slowly, antibody-based measurements of AGT protein correlate poorly with depletion of AGT activity immediately after BG. Thus, biochemical activity measurements remain the appropriate monitor of AGT during therapeutic modulation. These data provide the first and conclusive evidence of differential degradation rates of inactivated AGT in PBMCs and tumors of patients after treatment with BG and suggest that immunoreactive AGT measurements in PBMCs are a poor surrogate for AGT activity in tumor tissue.

Biopsy↗
PubMed2002

HHV-8 DNA in blood and the development of HIV-associated Kaposi's sarcoma in the era of HAART--a prospective evaluation.

OBJECTIVE: To explore the significance of HHV-8 viremia in HIV-positive individuals for the risk of developing Kaposi's sarcoma (KS) in the era of highly active antiretroviral therapy. METHODS: 237 HIV-positive patients were included in this prospective evaluation and followed over an average duration of 34 months. HHV-8 DNA in peripheral blood mononuclear cells (PBMCs) and CD4-lymphocytes were determined. In addition AIDS-defining conditions and antiretroviral therapy were documented of all participating subjects. RESULTS: HHV-8 DNA was detectable in PBMCs of 12.6% out of all individuals. 53.3% of these patients initially complained about KS, although 9.2% of patients without HHV-8 DNA in PBMCs were found on KS as well. Furthermore, four patients in total were observed with newly developed KS during follow up visits. None of these patients were noted with detectable HHV-8 DNA at their initial evaluation. CONCLUSIONS: Prevalence of HHV-8 DNA in PBMCs of subjects in this investigation was quite similar to former investigations. However, new diagnosed KS occurred less frequently than demonstrated in previous studies. All of those observed patients with new KS manifestations were negative for HHV-8 DNA in PBMCs at study entry. This observation differs from earlier studies which have postulated the detection of HHV-8 DNA in PBMCs as a predictive value for development of KS. Due to results as presented, a single HHV-8 DNA test in blood has no predictive value in support of predictability of KS development. With respect toto costs and to a less complicated performance antibody assays should be preferred.

Adult↗