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PubMed2001

"Bystander" amplification of PBMC cytokine responses to seasonal allergen in polysensitized atopic children.

BACKGROUND: Atopic children show increased expression and production of the Th2-associated cytokines IL-4, IL-5, IL-13, and IL-9 from PBMCs after stimulation with allergen, but it has previously not been clearly determined whether the Th2-cytokine production is restricted to the inhalant allergen the child is sensitized to, and whether perennial or seasonal allergens induce different cytokine responses. Our purpose was to determine whether in vitro Th2 cytokine production is specific to the sensitizing allergen, and to compare the cytokine responses to a perennial and a seasonal allergen in monosensitized and polysensitized children. METHODS: Using semiquantitative RT-PCR, we analyzed the expression of the cytokines IL-4, IL-5, IL-13, IL-9, IL-10, and IFN-gamma after stimulation of PBMCs with house-dust-mite (HDM) or ryegrass allergen. The cells were sampled from groups of 6-year-old children sensitized to either HDM (n=20) or ryegrass (n=24), or to both allergens (n=20), as well as from a nonatopic group (n=20). RESULTS: After stimulation with HDM allergen, PBMCs from children sensitized only to HDM expressed increased mRNA levels of the Th2 cytokines, but not of IL-10 and IFN-gamma, whereas ryegrass stimulation did not result in increased cytokine expression. PBMCs from children sensitized to HDM and ryegrass expressed increased Th2 cytokines after stimulation with either of the two allergens. In contrast, PBMCs from children sensitized only to ryegrass did not express increased levels after stimulation with either of the allergens. CONCLUSIONS: The expression of Th2 cytokines after in vitro stimulation of PBMCs from atopic children is specific to the sensitizing allergen, indicating that atopic status per se does not affect the type of T-cell response. In addition, T cells specific to seasonal allergens circulate in the blood out of season only if the child is concomitantly sensitized to a perennial allergen.

Allergens↗
PubMed2004

Conformational variation between allelic variants of cell-surface ovine prion protein.

The distribution of prion infectivity and PrPSc between peripheral lymphoid tissues suggests their possible haematogenic spread during the progression of natural scrapie in susceptible sheep. Since ovine PBMCs (peripheral blood mononuclear cells) express PrPC, they have the potential to carry or harbour disease-associated forms of PrP. To detect the possible presence of disease-associated PrP on the surface of blood cells, an understanding is required of the conformations that normal ovine cell-surface PrPC may adopt. In the present study, we have used monoclonal antibodies that recognize epitopes in either the N- or C-terminal portions of PrP to probe the conformations of PrPC on ovine PBMCs by flow cytometry. Although PBMCs from scrapie-susceptible and -resistant genotypes of sheep expressed similar levels of cell-surface PrPC, as judged by their reactivity with N-terminal-specific anti-PrP monoclonal antibodies, there was considerable genotypic heterogeneity in the region between helix-1 and residue 171. Cells from PrP-VRQ (V136R154Q171) sheep showed uniform reactivity with monoclonal antibodies that bound to epitopes around helix-1, whereas cells from PrP-ARQ (A136R154Q171) and PrP-ARR (A136R154R171) sheep showed variable binding. The region between b-strand-2 and residue 171, which includes a YYR motif, was buried or obscured in cell-surface PrPC on PBMCs from scrapie-susceptible and -resistant sheep. However, an epitope of PrPC that is influenced by residue 171 was more exposed on PBMCs from PrP-VRQ sheep than on PBMCs from the PrP-ARQ genotype. Our results highlight conformational variation between scrapie-susceptible and -resistant forms of cell-surface PrPC and also between allelic variants of susceptible genotypes.

Alleles↗
PubMed1998

Serum IgE level is negatively correlated with the ability of peripheral mononuclear cells to produce interferon gamma (IFNgamma): evidence of reduced expression of IFNgamma mRNA in atopic patients.

BACKGROUND: In patients with atopic diseases such as bronchial asthma and atopic dermatitis, an elevated serum IgE level is common. Several studies showed that interleukin-4(IL-4) and interferon-gamma (IFNgamma) are important for regulation of IgE production. OBJECTIVES: The study was designed to examine the pathogenesis of an elevated serum IgE level at the production levels of TH1-type and TH2-type cytokines. METHODS: The production of interleukin-2 (IL-2), IL-4, interleukin-5 (IL-5) and IFNgamma by peripheral blood mononuclear cells (PBMCs) stimulated with phytohemagglutinin (PHA) was analysed in 20 individuals with various serum IgE levels. The amount of IFNgamma mRNA in the stimulated PBMCs was analysed using a quantitative polymerase chain reaction method. RESULTS: Cytokine production was analysed simply as a function of serum IgE level. The serum IgE level was negatively correlated with the amount of IFNgamma secreted by PBMCs (n = 20, R = -0.724, P<0.005) when logarithmically transformed data were analysed, but were not or were only weakly correlated with those of IL-4, IL-2, or IL-5 secreted by these cells (n = 20). For investigation of the cause of reduced IFNgamma production in individuals whose serum IgE level are high, the amount of IFNgamma mRNA was determined. The levels of IFNgamma mRNA expression in the stimulated PBMCs correlated well with the amount of IFNgamma secreted by the PBMCs (n = 8, R = 0.947, P < 0.001). CONCLUSIONS: Reduced IFNgamma production ability due to reduced IFNgamma mRNA expression in PBMCs is associated with an elevated serum IgE level in atopic patients.

Adolescent↗
PubMed1998

The IGF-I/IGFBP system in congenital partial lipodystrophy.

BACKGROUND AND OBJECTIVES: Insulin and IGF-I interact at many levels. Little is known about the insulin-like growth factor-I/insulin-like growth factor binding proteins (IGF-I/IGFBP) system in congenital partial lipodystrophy, a syndrome characterized by insulin resistance, hyperinsulinaemia and absence of truncal and limb fat. Some cases have acromegaloid features with thick skin and large hands and feet in association with normal levels of circulating growth hormone. METHODS: In four females known with congenital partial lipodystrophy, hyperinsulinaemia with acromegaloid features, the number and affinity of the IGF-I receptors on peripheral blood mononuclear cells (PBMCs), and the concentration of circulating insulin, total and free IGF-I, IGFBP-1 and IGFBP-3 levels were measured in the fasting and the fed state. Cultures of PBMCs of the patients with lipodystrophy were also used to study the effect of IGF-I stimulation on thymidine uptake in vitro. MEASUREMENTS: In the subjects with lipodystrophy the affinity and the number of the IGF-I receptors on peripheral mononuclear cells (PBMCs) and erythrocytes did not differ significantly from controls in the fasting state. Insulin levels were significantly higher in subjects with lipodystrophy both in the fasting as well in the fed state. Total IGF-I, free IGF-I and IGFBP-3 levels did not differ but serum IGFBP-1 levels were lower in lipodystrophy subjects than in healthy controls. The free IGF-I/IGFBP-1 ratio was increased in lipodystrophy subjects both in the fasting and the fed states. The effects of IGF-I stimulation on thymidine uptake by PBMCs of lipodystrophy subjects in the absence of IGFBP-1 were not different from healthy controls cultures in vitro. When a combination of IGFBP-1 (in a concentration comparable to the fasting serum IGFBP-1 levels in lipodystrophy patients found in our study) and IGF-I was added to PBMC cultures from lipodystrophy patients no decrease in thymidine uptake by PBMCs was found. CONCLUSIONS: In the four subjects with lipodystrophy hyperinsulinaemia, lowered free IGF-I and IGFBP-1 levels, but increased free IGF-I/IGBP-1 ratios were observed. Low IGFBP-1 concentrations in culture media did not reduce the stimulating IGF-I effect on thymidine uptake by PBMCs from lipodystrophy patients. Our data suggest that the observed increased IGF-I/IGFBP-1 ratio in lipodystrophy patients contributes to an unopposed biological effect of IGF-I on IGF-I receptors, thereby inducing the development of acromegaloid features, acanthosis nigricans and polycystic ovaries in some patients with congenital partial lipodystrophy.

Adult↗
PubMed2003

Quantitative analysis of human mitochondrial DNA using a real-time PCR assay.

OBJECTIVES: Known for their ability to inhibit the human DNA polymerase-gamma, nucleoside analogues induce toxic effects on mitochondria ranging from increased serum lactate levels to fatal lactic acidosis. DNA polymerase-gamma ensures the mitochondrial DNA (mtDNA) replication and, thus, its inhibition leads to the decrease of the mtDNA. We describe a real-time PCR assay for mtDNA quantification associating DNA extraction procedures applied on peripheral blood mononuclear cells (PBMCs) and subcutaneous adipose tissues and to study the antiretroviral effect on mitochondria. METHODS: Total DNA was extracted from PBMCs and subcutaneous adipose tissues. Nuclear and mitochondrial genes were amplified to determine the number of copies of mtDNA per cell using a cyt-b recombinant plasmid as standard control. We analysed eight HIV-infected asymptomatic patients never treated, four patients who had been treated for 6 months with highly active antiretroviral therapy (HAART) and six non-infected donors. RESULTS: The mtDNA quantification gave rise to reproducible results as the mean coefficients of variation were 1.09% for replicates of samples undertaken 10 times within the same run, and 5.78% and 3.7% for replicates tested in five different runs at 1:100 and 1:1000 dilutions, respectively. Median levels of mtDNA in PBMCs of healthy donors, naive and treated HIV-infected patients were 2.94, 2.78 and 1.93 log HIV-1 RNA copies/mL, respectively. Whereas DNA from PBMCs was shown to be devoid of inhibitors, subcutaneous adipose tissues needed an extra treatment as they were found to be highly inhibited. CONCLUSIONS: The method generated consistent and reproducible results and was successfully applied to DNAs extracted from PBMCs and subcutaneous adipose tissues with adapted extraction. The mtDNA changes in PBMCs were found to be fast as they fall off after 6 months' therapy, decreasing from 2.78 to 1.93 log copies/mL.

Adipose Tissue↗
PubMed1999

GBV-C/HGV-RNA in serum and peripheral blood mononuclear cells in hemodialysis patients.

BACKGROUND: Hemodialysis patients are at high risk of hepatitis B, C, and G virus infection. The prevalence of GBV-C/HGV-RNA was analyzed in serum and peripheral blood mononuclear cells (PBMCs) from 52 hemodialysis patients. METHODS: GBV-C/HGV-RNA detection was performed by reverse transcription-polymerase chain reaction (RT-PCR) with primers of 5'-noncoding (5'-NC) and NS3 regions of the GBV-C/HGV genome. To increase sensitivity, serum samples were ultracentrifuged prior to the RT-PCR to concentrate the viral particles. The amplified products from 20 serum and 5 peripheral blood mononuclear cells (PBMC) samples were sequenced. RESULTS: GBV-C/HGV-RNA was detected in sera of 9 (17%) and in PBMCs of 30 (58%) patients. After serum ultracentrifugation, GBV-C/HGV-RNA was positive in 20 (95%) of the patients, with GBV-C/HGV-RNA only in PBMCs. Thus, GBV-C/HGV-RNA was detected in serum and PBMCs from 29 (56%) patients, four of whom had antibodies against GBV-C/HGV E2 protein (anti-HGE2); one patient (2%) had GBV-C/HGV-RNA only in PBMCs, but was anti-HGE2 positive. Seven (32%) patients who did not have GBV-C/HGV-RNA were anti-HGE2 positive. The nucleotide sequence homology between serum samples from the patients who were GBV-C/HGV-RNA positive after ultracentrifugation, and paired serum and PBMCs from five of them, ranged from 90 to 96% and from 92 to 98%, respectively. CONCLUSIONS: We found a high prevalence of GBV-C/HGV-RNA in serum and PBMC samples from hemodialysis patients. Whether or not this finding can be extended to other populations requires further study.

Aged↗
PubMed1998

Effect of glucose concentration, osmolality, and sterilization process of peritoneal dialysis fluids on cytokine production by peripheral blood mononuclear cells and polymorphonuclear cell functions in vitro.

We sought to investigate the effects of high glucose concentration, osmolality, and heat sterilization of peritoneal dialysis fluids on tumor necrosis factor-alpha (TNF-alpha) production by peripheral blood mononuclear cells (PBMC) and polymorphonuclear cell (PMN) functions. Blood samples were obtained from eight healthy volunteers. PBMCs and PMNs were harvested by centrifugation with Ficoll-Hypaque (Sigma, St Louis, MO). PBMC were incubated with an equal volume of test fluids and RPMI for 4 hours (pH equilibrated), followed by incubation for 20 hours in RPMI with or without endotoxin (10 ng/mL). Total TNF-alpha production was measured by radioimmunoassay. PMNs were incubated with pH-adjusted test fluids for 30 minutes. After incubation, phagocytosis was determined by the uptake of 14C-labeled Staphylococcus aureus, oxidative burst by reduction of ferricytochrome C to ferrouscytochrome C on stimulation with phorbol myristate acetate, and enzyme release by measurement of endotoxin-stimulated bactericidal/permeability increasing factor. To study the effects of increasing glucose concentration and osmolality on PBMC and PMN functions, we compared conventional 1.5% Dianeal (1.5%D), (Baxter Healthcare Corp, Deerfield, IL) 2.5% Dianeal (2.5%D), 4.25% Dianeal (4.25%D), and control (RPMI for PBMCs and Hank's balanced salt solution for PMNs). PMNs exposed to 4.25%D exhibited an inhibition of phagocytosis, phorbol myristate acetate (PMA)-stimulated oxidative burst, and bactericidal/permeability increasing factor release compared with control, 1.5%D, or 2.5%D. To study the effects of increased osmolality when controlled for glucose concentration, we compared 1.5%D with 1.5%D in which osmolality was increased to that of 4.25%D with the addition of either sodium chloride (1.5%D+NaCl) or mannitol (1.5%D+M). High osmolality induced higher TNF-alpha production by unstimulated PBMCs and decreased TNF-alpha production by endotoxin-stimulated PBMCs. PMN functions were also inhibited by high osmolality. To study the effects of increased glucose concentration when controlled for osmolality, we compared 4.25%D with 1.5%D+NaCl and 1.5%D+M. High glucose concentration induced an increase in TNF-alpha production by unstimulated PBMCs, a decrease in TNF-alpha production by endotoxin-stimulated PBMCs, and an inhibition of PMN functions. Finally, to investigate the effects of heat sterilization, we compared 4.25%D (heat sterilized) to a filter-sterilized 4.25%D (4.25%D-F). The filter-sterilized fluid induced less changes in PBMC and PMN functions compared with the heat-sterilized fluid. These data suggest that the high glucose concentration, high osmolality, and heat sterilization of peritoneal dialysis fluids adversely affect PBMC and PMN functions. These effects could predispose continuous ambulatory peritoneal dialysis patients to peritonitis, compromise host defense during infection, and jeopardize long-term survival of the peritoneal membrane.

Biocompatible Materials↗
PubMed1992

Prolactin synthesized and secreted by human peripheral blood mononuclear cells: an autocrine growth factor for lymphoproliferation.

Prolactin has been shown to have an immunoregulatory role in the rodent immune response. A prolactin-like molecule has also been found in mouse splenocytes and a human B-lymphoblastoid cell line. We have evaluated whether human peripheral blood mononuclear cells (PBMCs) synthesize and/or secrete prolactin. We used the polymerase chain reaction (PCR) to generate a 276-base-pair prolactin product from human PBMCs, and Southern blot analysis confirmed that it was related to prolactin. Western blotting using a polyclonal antibody to prolactin indicated that cell extracts prepared from human PBMCs contained a high molecular mass (60-kDa) immunoreactive prolactin. To determine whether this PBMC prolactin was being secreted, we developed a highly sensitive and specific hormonal enzyme-linked immunoplaque assay. With this assay, we were able to detect human prolactin secretion from concanavalin A (Con A)- or phytohemagglutinin-stimulated PBMCs but not from unstimulated PBMCs. We next sought to determine whether this secreted prolactin could function as an autocrine growth factor in lymphoproliferation. We observed that anti-human prolactin antiserum significantly inhibited human PBMC proliferation in response to Con A or phytohemagglutinin. We conclude that a prolactin-like molecule is synthesized and secreted by human PBMCs and that it functions in an autocrine manner as a growth factor for lymphoproliferation.

Base Sequence↗
PubMed1999

Gene expression, synthesis, and secretion of interleukin 18 and interleukin 1beta are differentially regulated in human blood mononuclear cells and mouse spleen cells.

Interleukin (IL)-18, formerly called interferon gamma (IFN-gamma)-inducing factor, is biologically and structurally related to IL-1beta. A comparison of gene expression, synthesis, and processing of IL-18 with that of IL-1beta was made in human peripheral blood mononuclear cells (PBMCs) and in human whole blood. Similar to IL-1beta, the precursor for IL-18 requires processing by caspase 1. In PBMCs, mature but not precursor IL-18 induces IFN-gamma; in whole human blood stimulated with endotoxin, inhibition of caspase 1 reduces IFN-gamma production by an IL-1beta-independent mechanism. Unlike the precursor for IL-1beta, precursor for IL-18 was expressed constitutively in PBMCs and in fresh whole blood from healthy human donors. Western blotting of endotoxin-stimulated PBMCs revealed processed IL-1beta in the supernatants via an caspase 1-dependent pathway. However, in the same supernatants, only unprocessed precursor IL-18 was found. Unexpectedly, precursor IL-18 was found in freshly obtained PBMCs and constitutive IL-18 gene expression was present in whole blood of healthy donors, whereas constitutive IL-1beta gene expression is absent. Similar to human PBMCs, mouse spleen cells also constitutively contained the preformed precursor for IL-18 and expressed steady-state IL-18 mRNA, but there was no IL-1beta protein and no spontaneous gene expression for IL-1beta in these same preparations. We conclude that although IL-18 and IL-1beta are likely members of the same family, constitutive gene expression, synthesis, and processing are different for the two cytokines.

Animals↗
PubMedDate not supplied

Cytokine profile of patients with mycosis fungoides and the immunomodulatory effect of AS101.

Cytokines are known to play a major role in the pathogenesis of mycosis fungoides, a cutaneous malignant neoplasm of CD 4 T cells. In the present study, we investigated the effect of AS101, a tellurium-based compound with immunomodulating properties, on the pattern of lymphokine production by peripheral blood mononuclear cells (PBMCs) from patients with mycosis fungoides. PBMCs were isolated from 35 patients with mycosis fungoides stage IA and IB before initiation of treatment and from 20 healthy sex and age-matched controls. Unstimulated and phytohaemagglutinin-stimulated PBMCs were tested with and without the addition of AS101. The production of interferon-gamma, interleukin 2 (IL-2), IL-2 receptor (IL-2R), interleukin 5 (IL-5) and interleukin 10 (IL-10) was determined by enzyme-linked immunosorbent assays. The effects of AS-101 on mycosis fungoides PBMCs were compared to those of healthy donor PBMCs. Significantly higher levels of IL-2R, IL-5 and IL-10 and significantly lower levels of interferon-gamma were found in the patients compared to the controls. There was no significant difference between the groups in the production of IL-2. AS101 inhibited the production of IL-2R, IL-5 and IL-10 and induced a significant increase in IL-2 levels in the mycosis fungoides PBMCs. These findings may have important clinical implications for the possible therapeutic benefit of AS101 in mycosis fungoides.

Adjuvants, Immunologic↗
PubMed2005

Dexamethasone influences human clock gene expression in bronchial epithelium and peripheral blood mononuclear cells in vitro.

We determined whether human peripheral blood mononuclear cells (PBMCs) could be used to analyze clock genes by studying their mRNA expressions in human bronchial epithelium (BEAS-2B) and PBMCs following stimulation by the glucocorticoid homologue dexamethasone (DEX) in vitro. PBMCs were obtained at 10:00 h from two diurnally active (approximately 07:00 to 23:00 h) healthy volunteers and were evaluated for hPer1 mRNA expression following DEX stimulation in vitro using real time-PCR analysis. DEX stimulation of human BEAS-2B cells and PBMCs in vitro led to a remarkable increase of hPer1 mRNA. The glucocorticoid rapidly affected the expression of hPer1 mRNA in PBMCs, suggesting that human PBMCs may be a useful surrogate marker for the investigation of drug effects on clock genes.

Adult↗
PubMed2004

Higher human T lymphotropic virus (HTLV) provirus load is associated with HTLV-I versus HTLV-II, with HTLV-II subtype A versus B, and with male sex and a history of blood transfusion.

BACKGROUND: High human T lymphotropic virus (HTLV)-I provirus load (VL) has been associated with an increased risk of HTLV-associated myelopathy, but little is known about variation in HTLV-I or -II VLs by demographic characteristics and risk behaviors. METHODS: We measured HTLV-I and HTLV-II VLs in a large cohort of 127 HTLV-I-seropositive and 328 HTLV-II-seropositive former blood donors, by use of real-time polymerase chain reaction using tax primers. Multivariable linear regression was used to control for confounding by relevant covariates. RESULTS: The mean VLs were 3.28 log(10) copies/10(6) peripheral blood mononuclear cells (PBMCs) (range, 0.5-5.3 log(10) copies/10(6) PBMCs) for HTLV-I and 2.60 log(10) copies/10(6) PBMCs (range, 0.05-5.95 log(10) copies/10(6) PBMCs) for HTLV-II (P<.0001). HTLV-II VLs were higher in those subjects with subtype A infection (mean, 2.82 log(10) copies/10(6) PBMCs) than in those with subtype B infection (mean, 2.29 log(10) copies/10(6) PBMCs) (P=.005). Higher HTLV-I VL was associated with previous receipt of a blood transfusion (P=.04), and lower HTLV-II VL was associated with female sex (P=.007). These associations persisted in virus-specific multivariate linear regression models controlling for potential confounding variables. CONCLUSIONS: VL was significantly higher in HTLV-I than in HTLV-II infection and was higher in HTLV-II subtype A than in HTLV-II subtype B infection. Chronic HTLV VLs may be related to the infectious dose acquired at the time of infection, with higher VLs following acquisition by blood transfusion and lower VLs following sexual acquisition.

Adult↗
PubMed2004

Cellular viral rebound after cessation of potent antiretroviral therapy predicted by levels of multiply spliced HIV-1 RNA encoding nef.

To characterize newly arising replication of human immunodeficiency virus (HIV) type 1 in vivo at the cellular level, distinct viral RNA species in peripheral blood mononuclear cells (PBMCs) from HIV-1-infected patients were monitored during 2 weeks of structured treatment interruption (STI). HIV-1 RNA encoding tat/rev and PBMC-associated virions were almost completely depleted during antiretroviral therapy and emerged simultaneously after 2 weeks of STI, thus specifically reflecting productive viral infection at the cellular level. The magnitude of these correlates of reappearing cellular viral replication was predicted by during-therapy levels of nef transcripts in PBMCs. Significant rebound of plasma viremia, representing the progeny of a broader range of anatomical compartments, preceded and predicted productive infection in PBMCs. Thus, cellular viral rebound in PBMCs likely was primed before STI by the expression of nef in HIV-1-infected PBMCs that lacked virion production and was subsequently triggered by the plasma viremia that preceded the recurrence of productively infected PBMCs.

Anti-HIV Agents↗
PubMed2004

Corticoids normalize leukocyte production of macrophage migration inhibitory factor in septic shock.

BACKGROUND: A regulatory loop between macrophage migration inhibitory factor (MIF) and glucocorticoids has been characterized in animal models. Renewed interest in glucocorticoid treatment for septic shock offers an opportunity to analyze this regulatory loop in humans. METHODS: We investigated the ex vivo release of MIF by peripheral blood mononuclear cells (PBMCs) sampled from glucocorticoid-treated and -untreated patients with septic shock. Blood was obtained, before glucocorticoid treatment, and within the first day of treatment, from patients with septic shock who required treatment with moderate doses of hydrocortisone and fludrocortisone. RESULTS: PBMCs from patients contained significantly higher amounts of MIF than cells from healthy control subjects. In culture, spontaneous release of MIF and release induced by lipopolysaccharide (LPS), heat-killed staphylococci, and red blood cell lysates were significantly higher in patients than in control subjects. PBMCs from patients treated with glucocorticoids showed a lower release of MIF in response to LPS, heat-killed Escherichia coli, and peptidoglycan than did PBMCs from untreated patients and showed levels similar to PBMCs from healthy control subjects. CONCLUSION: To our knowledge, MIF is the first proinflammatory cytokine in which ex vivo release by circulating cells is enhanced during sepsis. Glucocorticoid treatment normalized the release of MIF by circulating PBMCs from patients with septic shock.

Aged↗
PubMed2005

CD8+ T cell depletion amplifies hepatitis C virus replication in peripheral blood mononuclear cells.

We investigated the ability of CD8+ T cells to inhibit hepatitis C virus (HCV) replication in peripheral blood mononuclear cells (PBMCs). PBMCs isolated from 11 of 20 HCV-infected subjects had no detectable HCV RNA. Removal of CD8+ T cells from these PBMCs resulted in detection of HCV RNA, and depletion of CD8+ T cells from PBMCs that had detectable HCV RNA amplified HCV replication. Reconstitution of CD8- PBMCs with autologous CD8+ T cells led to inhibition of HCV replication. Interferon-gamma produced by CD8+ T cells was partially responsible for CD8+ T cell-mediated noncytotoxic anti-HCV activity in PBMCs. This noncytotoxic anti-HCV activity was confirmed in HCV replicon cells. Supernatants from CD8+ T cell cultures inhibited HCV RNA expression in the replicon cells. These findings may have important implications for the immunopathogenesis of HCV in both immune and hepatic cells and are relevant to the development of host innate immunity-based anti-HCV interventions.

Antiviral Agents↗
PubMed2001

Effects of norepinephrine, HIV type 1 infection, and leukocyte interactions with endothelial cells on the expression of matrix metalloproteinases.

The expression of matrix metalloproteinases (MMPs) associated with AIDS-related cardiomypathies and cocaine abuse was examined in an in vitro coculture model. Human peripheral blood mononuclear cells (PBMCs), HIV infected or uninfected, were placed in coculture with primary human cardiac microvascular endothelial cells (HMVEC-C) in the presence or absence of the cocaine-inducible catecholamine norepinephrine (NE). Culture supernatants were assayed for MMP-1, -2, -3, -7, -9, and -13, and for tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-2, by enzyme-linked immunosorbent assay. Low levels of constitutively expressed MMP-1 and -2 were detected in individual cultures of HMVEC-C and PBMCs. NE did not induce MMP or TIMP expression by HMVEC-C and caused modest increases (3- to 4-fold) in MMP-1 and -2 by uninfected PBMCs. Increased levels of NE-induced MMP-1 (5-fold) and MMP -2 (15-fold) were detected in cocultures of HMVEC-C and uninfected PBMCs. HIV infection enhanced MMP-1 (46-fold) and MMP-2 (48-fold) and active MMP-7 (33-fold) and MMP-9 (50-fold) by PBMCs. Coculture of HIV-infected PBMCs with HMVEC-C increased MMP-1 (110-fold) and MMP-2 (307-fold) but not active MMP-7 and -9. The combination of NE, HIV infection, and coculture increased MMP-1 (126-fold) and MMP-2 (467-fold), and active MMP-7 (65-fold) and MMP-9 (75-fold). MMP-3 or-13 was not detected in any of the treatment groups and TIMP-1 and -2 appeared inversely proportional to the observed levels of MMPs. These results suggest that HIV infection, NE, and leukocyte endothelial interactions demonstrate separate and overlapping cooperative effects on the regulation of expression of TIMPs and MMPs associated with AIDS-related cardiomyopathies.

Cardiomyopathies↗
PubMed2006

Peripheral blood mononuclear and tumor cell pharmacodynamics of the novel epothilone B analogue, ixabepilone.

BACKGROUND: We previously demonstrated that peak microtubule bundle formation (MBF) in peripheral blood mononuclear cells (PBMCs) occurs at the end of drug infusion and correlates with drug pharmacokinetics (PK). In the current study, a new expanded evaluation of drug target effect was undertaken. PATIENTS AND METHODS: Patients with advanced solid malignancies were treated with ixabepilone 40 mg/m2 administered as a 1-h i.v. infusion every 3 weeks. Blood, plasma, and tumor tissue sampling was carried out to characterize pharmacodynamics and PK. RESULTS: Forty-seven patients were treated with 141 cycles of ixabepilone. In both PBMCs (n=27) and tumor cells (n=9), peak MBF occurred at the end of infusion; however, at 24-72 h after drug infusion, the number of cells with MBF was significantly greater in tumor cells, relative to PBMCs. A Hill model (EC50=109.65 ng/ml; r2=0.94) was fitted, which demonstrated a relationship between percentage of PBMCs with MBF and plasma ixabepilone concentration. The percentage of PBMCs with MBF at the end of infusion also correlated with severity of neutropenia (P=0.050). CONCLUSIONS: Plasma ixabepilone concentration and severity of neutropenia correlate with the level of MBF in PBMCs. Therefore, this technically straightforward assay should be considered as a complement to the clinical development of novel microtubule-binding agents.

Adult↗
PubMed1988

Newcastle disease virus as an antineoplastic agent: induction of tumor necrosis factor-alpha and augmentation of its cytotoxicity.

The oncolytic strain 73-T of Newcastle disease virus (NDV) has been reported to be beneficial in the treatment of cancer patients, but little is known about its mechanism of action. In this study, NDV strain 73-T and a wild-type isolate of NDV were found to be potent inducers of tumor necrosis factor (TNF) production by both human peripheral blood mononuclear cells (PBMCs) and rat splenocytes. Antibody inhibition experiments identified TNF-alpha as the major species of TNF induced by NDV in PBMCs. The effect of recombinant human TNF-alpha (rHuTNF-alpha) on human cancer cells was then examined. Neither rHuTNF-alpha nor supernatants from NDV-stimulated PBMCs were cytotoxic toward the TNF-resistant human malignant melanoma cell line MEL-14. However, when MEL-14 cells were treated with NDV strain 73-T, both rHuTNF-alpha and supernatants from NDV-stimulated PBMCs killed 48% and 55%, respectively, of these tumor cells. Treatment with NDV also conferred TNF susceptibility to the TNF-resistant human malignant melanoma cell line MEL-21 and the human myelogenous leukemia cell line K562. In contrast to its enhanced cytotoxicity toward NDV-treated cancer cells, rHuTNF-alpha had no effect on NDV-treated normal human PBMCs proliferating in response to concanavalin A. These results suggest two important mechanisms for the antineoplastic activity of NDV: (a) induction of TNF-alpha secretion by human PBMCs and (b) enhancement of the sensitivity of neoplastic cells to the cytolytic effects of TNF-alpha.

Antibodies↗