Cytoskeleton of human mononuclear cells as a possible peripheral marker for phenylalanine neurotoxicity in PKU

In this work we tested human mononuclear cells as a peripheral marker to study neurotoxicity of phenylalanine (Phe). Slices of cerebral cortex of rats or human mononuclear cells were incubated with different concentrations of Phe and/or Ala in the presence of ³²P-orthophosphate, the cytoskeletal fraction was extracted, and the radioactivity incorporated into intermediate filament proteins was measured. Our results show that 2 mM Phe as well as 1 mM Ala are effective in increasing the ³²P in vitro incorporation into IFs in both tissues. When cerebral cortex slices or mononuclear cells were incubated with different concentrations of Phe and/or Ala, the effects on the ³²P in vitro incorporation into IF proteins was compatible with an antagonistic mechanism of action of the two amino acids on the enzymes of the phosphorylating system. In addition, these blood cells may be a possible peripheral marker to study neurotoxicity of Phe in patients with PKU.     

Mineralocorticoid effector mechanism in human mononuclear leukocytes

Mineralocorticoid receptors and mineralocorticoid effector mechanism were determined in mononuclear leukocytes (MNL) from normal subjects. The hierarchy of affinities of competitors for the receptor was similar to that described in other non-classical target tissues for aldosterone. In spite of the relative high affinity of cortisol for the receptor, these binding sites are occupied in vivo by aldosterone and play a mineralocorticoid effect in terms of electrolyte content of the cells. The effect of aldosterone is to prevent the loss of electrolytes due to incubation in medium alone and this action is reversed by addition of actinomycin D. In addition, the incubation of the MNL with aldosterone plus human alpha ANP leads to complete block of the action of aldosterone alone. This effect is not mediated by binding of alpha ANP to mineralocorticoid receptors but is probably related to a some postereceptorial effect of aldosterone at the level of plasma membrane. We conclude that the model of MNL is a good tool for studying mineralocorticoid receptors regulation and consequent effector mechanism in humans.     

Glycan recognition by human blood mononuclear cells with an emphasis on dendritic cells

Dendritic cells (DCs) play crucial roles in innate and adaptive immune response, for which reason targeting antigen to these cells is an important strategy for improvement of vaccine development. To this end, we explored recognition of DCs lectins by glycans. For selection of the glycan “vector”, a library of 229 fluorescent glycoprobes was employed to assess interaction with the CD14^low/-CD16⁺CD83⁺ blood mononuclear cell population containing the DCs known for their importance in antigen presentation to T-lymphocytes. It was found that: 1) the glycan-binding profiles of this CD14^low/-CD16⁺CD83⁺ subpopulation were similar but not identical to DCs of monocyte origin (moDCs); 2) the highest percentage of probe-positive cells in this CD14 ^low/-CD16⁺CD83⁺ subpopulation was observed for GalNAcα1-2Galβ (A_di), (Neu5Acα)₃ and three mannose-reach glycans; 3) subpopulation of CD14^low/-CD16⁺ cells preferentially bound 4’-O-Su-LacdiNAc. Considering the published data on specificity of DCs binding, the glycans showing particular selectivity for the CD14 ^low/-CD16⁺CD83⁺ cells are likely interacting with macrophage galactose binding lectin (MGL), siglec-7 and dectin-2. In contrast, DC-SIGN is not apparently involved, even in case of mannose-rich glycans. Taking into consideration potential in vivo competition between glycan “vectors” and glycans within glycocalyx, attempting to target vaccine to DCs glycan-binding receptors should focus on A_di and (Neu5Acα)₃ as the most promising vectors.     

Antibacterial activity of human mononuclear leukocytes againstStaphylococcus aureus

Human mononuclear leukocytes killStaphylococcus aureus cellsin vitro. The killing of the bacteria takes place even in the absence of antibodies. The presence of antibodies (in an autologous inactivated serum) usually enhances the antibacterial activity of mononuclear leukocytes. In some cases, however, this activity is markedly decreased by the serum, probably depending of the spectrum of antibodies contained in the serum. The antibacterial activity of mononuclear leukocytes is mostly due to monocytes because their depletion causes substantial drop or the activity disappearance. We failed to demonstrate in the case ofS. aureus the antibacterial cytotoxicity of T lymphocytes described by some authors dealing with Gram-negative bacteria. Large differences in the structure of the bacterial cell wall underlie apparently the different sensitivity of G⁺ and G⁻ bacteria to some protective mechanisms of the host. In the antibacterial assay againstS. aureus, electron microscopy revealed a maximal activation of monocytes which phagocytized the bacteria although extracellular killing is not excluded. Electronoptical findings point also to a possible participation of NK cells in the antibacterial cytotoxicity againstS. aureus.     

Parathyroid hormone receptors in circulating human mononuclear leukocytes

In this article we demonstrate receptors for parathyroid hormone in circulating mononuclear leukocytes using the radioiodinated analogue (8,18 norleucine, 34 tyrosine) bPTH 1-34 (bovine parathyroid hormone 1-34). Specific binding, which is reversible and saturable, equilibrates within 5 min at 0-4 degrees C with a calculated KD of 8.9 X 10(-11) M. This binding has a pH maximum of 7.0, is magnesium-dependent, and is inversely related to medium calcium concentration. Such binding is completely inhibited by simultaneous addition of 4 ng/ml of bovine parathyroid hormone 1-34, 5 ng/ml of bovine parathyroid hormone 1-84, or 5 ng/ml (8,18 norleucine, 34 Tyr) of 3-34 bPTH, but is unaffected by a biologically inactive parathyroid hormone fragment or other unrelated peptide hormones. Cyclic AMP accumulation increases 3-fold after 5 min exposure of mononuclear leukocytes to bPTH 1-34 in concentrations as low as 1 X 10(-9) M. Lymphocytes appear to be the circulating cells which interact with PTH as indicated by the observations that: 1) lymphocyte-enriched preparations bind three times as much radioligand/cell as do mixed mononuclear leukocytes, 2) monocytes, platelets, granulocytes, and erythrocytes do not bind PTH, and 3) monocytes, but not lymphocytes, degrade the hormone.     

Interferon-mediated inhibition of prostaglandin synthesis in human mononuclear leukocytes

In this study, the question of whether human leukocyte-derived and fibroblast-derived interferon had an effect on prostaglandin metabolism in human peripheral blood mononuclear cells has been considered. Both leukocyte- and fibroblast-derived interferon were potent inhibitors of mononuclear cell prostaglandin synthesis at low physiological concentrations. Inhibition required a minimum incubation of 1 hr. Interferon had no effect on release of arachidonic acid; synthesis of hydroxy fatty acids was slightly increased.     

Interferon with novel characteristics produced by human mononuclear leukocytes

Treatment of human peripheral blood mononuclear leukocytes with phytohaemmaglutinin and the tumour promoter teleocidin, results in the production of large amounts of interferon-gamma and significant amounts of a novel interferon-like substance which we tentatively class as interferon-delta. This novel interferon type possesses all the important characteristics of classical interferon but, of various cell types tested, has antiviral activity only in trisomy-21 human fibroblasts. It differs decisively from previously identified interferon types in its antigenic, biological and physicochemical properties.     

alpha-Interferon increases immunoglobulin production in cultured human mononuclear leukocytes

Interferons (IFN) are known to modulate immune responses in either an inhibitory or a stimulating manner. The present study was initiated to investigate the mechanisms by which alpha-IFN modulates Ig production of human peripheral blood mononuclear cells (PBMC). IgG and IgM production was measured in pokeweed mitogen- (PWM) stimulated 7-day cultures of PBMC. Significant enhancement of IgM and IgG production was observed when alpha-IFN was added. Overnight preincubation followed by washing also produced significant enhancement. The effect of alpha-IFN was not obtained in the absence of PWM or T cells. The effect of alpha-IFN on cultures of B and T cells was not altered by irradiation of T cells (2000 rad). alpha-IFN was not shown to enhance the production of helper factor but did increase the responsiveness of B cells to helper factor if the B cells were preincubated with alpha-IFN. Finally, alpha-IFN did not increase the Ig production of PBMC induced by Epstein Barr virus (EBV), and the outgrowth of EBV-infected PBMC was not affected. Overall, these results show for the first time that the effect of alpha-IFN on PBMC is due to an enhanced responsiveness of B cells to helper factors produced by radioresistant T cells.     

Harnessing human plasmacytoid dendritic cells as professional APCs

The plasmacytoid dendritic cell (pDC) constitutes a unique DC subset that links the innate and adaptive arm of the immune system. Whereas the unique capability of pDCs to produce large amounts of type I IFNs in response to pathogen recognition is generally accepted,their antigen-presenting function is often neglected since most studies on antigen presentation are aimed at other DC subsets. Recently, pDCs were demonstrated capable to present antigen leading to protective tumor immunity. In this review, we discuss how pDCs could be exploited in the fight against cancer by analyzing their capacity to capture,process and (cross-) present antigen.     

Glucose-6-phosphatase as a cytochemical marker of endoplasmic reticulum in human leukocytes and platelets

Leukocytes and platelets, freshly isolated from normal human blood, were tested cytochemically for glucose-6-phosphatase (G-6-Pase) by a modified Wachstein-Meisel method. The enzyme was present in the endoplasmic reticulum (ER) and perinuclear cisternae of all five types of leukocytes and in the ER of platelets. The reaction product from the cytochemical test distinguished the ER from other intracellular membrane-limited cisternae (i.e., the smooth pinocytic tubules of monocytes and the surface-connected canalicular system of platelets) and thus is a valuable marker of the ER. The cytochemical test also showed that the ER of polymorphonuclear leukocytes (PMN), usually obscured by abundant granules in cells prepared for morphological examination, is more extensive than formerly appreciated. This is the first demonstration of G-6-Pase in human leukocytes. Its precise role in leukocyte metabolism can now be investigated.     

Follicular dendritic cells emerge from ubiquitous perivascular precursors

The differentiation of follicular dendritic cells (FDC) is essential to the remarkable microanatomic plasticity of lymphoid follicles. Here we show that FDC arise from ubiquitous perivascular precursors (preFDC) expressing platelet-derived growth factor receptor β (PDGFRβ). PDGFRβ-Cre-driven reporter gene recombination resulted in FDC labeling, whereas conditional ablation of PDGFRβ(+)-derived cells abolished FDC, indicating that FDC originate from PDGFRβ(+) cells. Lymphotoxin-α-overexpressing prion protein (PrP)(+) kidneys developed PrP(+) FDC after transplantation into PrP(-) mice, confirming that preFDC exist outside lymphoid organs. Adipose tissue-derived PDGFRβ(+) stromal-vascular cells responded to FDC maturation factors and, when transplanted into lymphotoxin β receptor (LTβR)(-) kidney capsules, differentiated into Mfge8(+)CD21/35(+)FcγRIIβ(+)PrP(+) FDC capable of trapping immune complexes and recruiting B cells. Spleens of lymphocyte-deficient mice contained perivascular PDGFRβ(+) FDC precursors whose expansion required both lymphoid tissue inducer (LTi) cells and lymphotoxin. The ubiquity of preFDC and their strategic location at blood vessels may explain the de novo generation of organized lymphoid tissue at sites of lymphocytic inflammation.     

Preferential infection of dendritic cells during human immunodeficiency virus type 1 infection of blood leukocytes

Human immunodeficiency virus type 1 (HIV-1) transmission by the parenteral route is similar to mucosal transmission in the predominance of virus using the CCR5 coreceptor (R5 virus), but it is unclear whether blood dendritic cells (DCs), monocytes, or T cells are the cells initially infected. We used ex vivo HIV-1 infection of sorted blood mononuclear cells to model the in vivo infection of blood leukocytes. Using quantitative real-time PCR to detect full-length HIV-1 DNA, both sorted CD11c⁺ myeloid and CD11c⁻ plasmacytoid DCs were more frequently infected than other blood mononuclear cells, including CD16⁺ or CD14⁺ monocytes or resting CD4⁺ T cells. There was a strong correlation between CCR5 coreceptor use and preferential DC infection across a range of HIV-1 isolates. After infection of unsorted blood mononuclear cells, HIV-1 was initially detected in the CD11c⁺ DCs and later in other leukocytes, including clustering DCs and activated T cells. DC infection with R5 virus was productive, as shown by efficient transmission to CD4⁺ T cells in coculture. Blood DCs infected with HIV-1 in vitro and cultured alone expressed only low levels of multiply spliced HIV-1 RNA unless cocultured with CD4⁺ T cells. Early selective infection of immature blood DCs by R5 virus and upregulation of viral expression during DC-T-cell interaction and transmission provide a potential pathway for R5 selection following parenteral transmission.     

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. Download video file.^{(65M, mp4)}     

Origin,precursors and differentiation of mouse dendritic cells

Functional specialization allows defined dendritic-cell (DC) subsets to induce efficient defence mechanisms against pathogens and tumour cells, and maintain T-cell tolerance by inducing the inactivation of autoreactive T cells. A crucial question, which has important implications for both our understanding of the induction and control of immunity by DCs, as well as the use of DCs for immunotherapy, is whether the functional diversity of DCs results from the existence of developmentally independent DC subpopulations, or whether DC subsets that share a common differentiation origin acquire specific functions in response to environmental signals. This review discusses recent findings on mouse DC development.     

Metabolic syndrome and acute hyperglycemia are associated with endoplasmic reticulum stress in human mononuclear cells

Endoplasmic reticulum (ER) stress and the activation of the unfolded protein response (UPR) have been implicated in a number of complications associated with diabetes mellitus including micro‐ and macrovascular dysfunction. In this study we examine ER stress levels in blood cells isolated from human subjects with metabolic syndrome and in healthy controls. Total RNA and protein were isolated from leukocytes and the levels of specific ER stress markers were quantified by real‐time‐PCR and immunoblot analysis. Our results indicate that, compared to healthy controls, individuals with metabolic syndrome have elevated mRNA levels of genes indicative of ER stress; including spliced XBP‐1 (sXBP‐1), Grp78, and CHOP. Induced ER stress levels correlate with blood glucose but not plasma lipid concentration. Furthermore, in healthy individuals, a standard 75 g oral glucose challenge produced a significant elevation in spliced XBP‐1 (1.3 fold), Grp78 (2.0 fold), and calreticulin (3.5 fold) mRNA 60 min post challenge and a significant increase in Grp78 (2.0 fold), calreticulin (2.7 fold) protein levels 2 h postchallenge, relative to fasting levels. The UPR was also activated ex vivo, in human leukocytes cultured in the presence of 15 mmol/l glucose, supporting a specific role for glucose. The oral glucose challenge was associated with a significant increase in the expression of inflammatory cytokines, including interleukin (IL)‐1α/β, IL‐6, and IL‐8, that may result from ER stress. These findings suggest that there is an association between both acute and chronic dysglycemia and ER stress in humans.     

Identification of thetrans-stilbene oxide-active glutathione transferase in human mononuclear leukocytes and in liver as GST1

A glutathione transferase from human mononuclear leukocytes with a high activity towardtrans-stilbene oxide (GT-tSBO) has been studied in liver and blood from fetus and adults and in blood from neonates. Using starch gel electrophoresis, different phenotypes of GST1 have been determined, GST1 0, GST1 1, and GST1 2. As judged from activity measurements and the fact that only those individuals who express the null allele of GST1, the GST1 0, which has a low activity towardtrans-stilbene oxide, it is concluded that the hepatic transferase GST1 is identical to GT-tSBO, as well as to hepatic transferase μ. In addition, it has been shown that the different genotypes of GST1 1 (GST1 1-1, GST1 1-0) and GST1 2 (GST1 2-2, GST1 2-0) can be separated by measuring the GT-tSBO activity in whole blood from the same individual. It is also demonstrated that GT-tSBO activity is much lower in fetal liver, approximately 10 times, compared with adult liver, while this activity seems to be unchanged in the blood from fetus and adults, as well as in neonates.     

Arachidonic acid metabolism among human mononuclear leukocytes. Lipoxygenase-related pathways

Human peripheral blood mononuclear cells were isolated and assessed for the presence of contaminating polymorphonuclear leukocytes and platelets. Incubations of these cell isolates were performed in the presence or absence of the calcium ionophore A23187 and/or 1-14C-labeled or unlabeled arachidonic acid. Using reverse phase high pressure liquid chromatography with simultaneous monitoring of ultraviolet light absorption at 229 and 280 nm and, where appropriate, of radioactivity, our studies reveal that human peripheral blood mononuclear cells generate leukotrienes C4 and B4 (LTC4 and LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) following stimulation with A23187. The ratio of LTC4 to LTB4 was approximately 10-fold greater among the mononuclear cells than among similar incubations of polymorphonuclear leukocytes. Furthermore, the mononuclear cells failed to metabolize LTB4 into the omega-hydroxy or omega-carboxy derivatives that were always present in, and very characteristic of incubations of polymorphonuclear leukocytes. Depletion of monocytes from the mononuclear cells by double adherence resulted in virtual loss of the generation of 5-lipoxygenase-derived products by the remaining nonadherent cells, supporting the conclusion that the monocytes and not the lymphocytes were the source of LTC4, LTB4, and 5-HETE. The presence of both 12-HETE and the cyclooxygenase-derived 12-hydroxyheptadecatrienoic acid correlated with the degree of platelet contamination, suggesting that the platelets account for the presence of these compounds.     

Induction of interleukins by mycoplasma in culture of human mononuclear leukocytes

Mycoplasma shows a variety of effects on immune system, including the activation of macrophage, the increase in T cell cytotoxicity, and the enhancement of the proliferation and maturation of B cells, etc. As it is well known that many cytokines regulate the immune system, it is interesting to examine whether or not human peripheral blood mononuclear cells (PBMC) produce interleukin (IL) in response to mycoplasmas. In the present study, human PMBC were incubated with 7 species of mycoplasmas for 48 hours, and IL-1 beta, IL-2 and IL-6 activities in the supernatants were determined by ELISA. All the species of mycoplasmas were able to induce IL-1 beta and IL-6, although IL-2 was induced only by M. pneumoniae. These results suggest that the influence of mycoplasma infection on immune system may be partly due to the interleukins induced by mycoplasmas.     

Uptake of nickel(II) by human peripheral mononuclear leukocytes

Dithiocarbamate-mediated nickel(II) uptake by human peripheral mononuclear leukocytes (mostly lymphocytes) was examined. The lipophilic ligands diethyldithiocarbamate (DDC) and ammonium pyrrolidinedithiocarbamate (APDC) enhanced the cellular association of nickel(II), while ammonium dithiocarbamate (AD) had no effect. Sequential incubations (ligand first followed by nickel(II)) and concurrent experiments (simultaneous exposure) yielded similar results. Cell fractionation studies showed that DDC promoted cytosolic accumulation of nickel(II), rather than in the residual cell pellet. The observations reported are interpreted in terms of the "Equilibrium" model of metal-ion uptake by cells proposed by Professor Williams. Although nickel(II) transformed lymphocytes in vitro from an individual with dermal manifestation of nickel sensitivity to lymphoblasts, there was no apparent difference in nickel(II) uptake capacity over lymphocytes from nonsensitized controls.