Stromal-derived factor-1 in human tumors recruits and alters the function of plasmacytoid precursor dendritic cells.

Dendritic-cell (DC) trafficking and function in tumors is poorly characterized, with studies confined to myeloid DCs (DC1s). Tumors inhibit DC1 migration and function, likely hindering specific immunity. The role of plasmacytoid DCs (DC2s) in tumor immunity is unknown. We show here that malignant human ovarian epithelial tumor cells express very high levels of stromal-derived factor-1, which induces DC2 precursor (preDC2) chemotaxis and adhesion/transmigration, upregulates preDC2 very late antigen (VLA)-5, and protects preDC2s from tumor macrophage interleukin-10-induced apoptosis, all through CXC chemokine receptor-4. The VLA-5 ligand vascular-cell adhesion molecule-1 mediated preDC2 adhesion/transmigration. Tumor preDC2s induced significant T-cell interleukin-10 unrelated to preDC2 differentiation or activation state, and this contributed to poor T-cell activation. Myeloid precursor DCs (preDC1s) were not detected. Tumors may weaken immunity by attracting preDC2s and protecting them from the harsh microenvironment, and by altering preDC1 distribution.

Reciprocal regulation of plasmacytoid dendritic cells and monocytes during viral infection.

Reciprocal regulation of opposing functions characterizes biological systems. We now show that adenovirus-infected plasmacytoid dendritic cells (PDC) inhibit monocyte to myeloid dendritic cell (MDC) differentiation and function, and that adenovirus-infected monocytes inhibit PDC type I interferon secretion. Adenovirus-infected PDC secreted IFN-alpha, beta and omega in an 86:2:1 ratio. PDC type I interferons inhibited MDC differentiation and function (reduced IL-12 secretion, IFN-gamma induction, MLR and CD40 expression, and increased CD1a(+)CD14(+) cells). Type I interferon receptor blocking antibody reversed all PDC effects, and recombinant IFN-alpha, beta or omega replicated all effects, except reduced CD40. Adenovirus-infected monocytes suppressed PDC type I interferon secretion, which was reversed with anti-IL-10 neutralizing antibodies. Exogenous IL-10 suppressed PDC type I interferon secretion without reducing PDC viability. Therefore, monocyte IL-10 regulates PDC type I interferon secretion, and PDC type I interferons inhibit MDC differentiation and function. Such reciprocal regulation of potentially opposing influences may help modulate anti-pathogen immunity.

Macrophage-derived dendritic cells have strong Th1-polarizing potential mediated by beta-chemokines rather than IL-12.

Monocyte-derived dendritic cells (MDDCs) activate naive T lymphocytes to induce adaptive immunity, effecting Th1 polarization through IL-12. However, little is known about other potential DC Th1 polarizing mechanisms, or how T cell polarization may be affected by DCs differentiating in, or exposed to, a proinflammatory environment. Macrophages (MPhis) are DC precursors abundant in inflamed tissues, lymph nodes, and tumors. Thus we studied the T cell-activating and -polarizing properties of MPhi-derived DCs (PhiDCs). Monocytes were cultured in MPhi-CSF (M-CSF) to produce MPhis, which were then differentiated into DCs following culture with GM-CSF plus IL-4. PhiDCs activated a significant allogeneic MLR and were significantly better than MDDCs in activating T cells with superantigen. Most strikingly, PhiDCs elicited up to 9-fold more IFN-gamma from naive or Ag-specific T cells compared with MDDCs (with equivalent IL-4 secretion), despite producing up to 9-fold less IL-12. Neutralization of MDDC, but not PhiDC IL-12 significantly inhibited T cell IFN-gamma induction. PhiDCs produced up to 12-fold more beta-chemokines (macrophage-inflammatory protein-1alpha, -1beta, and RANTES) than MDDCs. Ab blockade of CCR5, but not CXC chemokine receptor 4, inhibited T cell IFN-gamma induction by PhiDCs significantly greater than by MDDCs. Thus DCs differentiating from MPhis induce T cell IFN-gamma through beta-chemokines with little or no requirement for IL-12. Myeloid DCs arising from distinct precursor cells may have differing properties, including different mechanisms of Th1 polarization. These data are the first reports of IFN-gamma induction through chemokines by DCs.

Functional expression of the MHC class I-related receptor, FcRn, in endothelial cells of mice.

Our recent data indicate that the MHC class I-related receptor, FcRn, plays a role in regulating serum IgG levels, in addition to its known role in transferring IgG from mother to young. In the current study, the distribution of FcRn in adult mice has been investigated using several approaches. First, tissue distribution of anti-FcRn F(ab')2, murine IgG1 and recombinant, IgG1-derived Fc-hinge fragments has been analyzed, and these FcRn binding proteins localize predominantly in skin and muscle with lesser amounts in liver and adipose tissue. Second, histochemical analyses of muscle and liver with anti-FcRn F(ab')2 indicate that FcRn is expressed in the endothelium of small arterioles and capillaries, but not in larger vessels such as the central vein and portal vasculature. Third, immunoprecipitation and immunofluorescence studies of cultured murine endothelial cells show that functional FcRn is expressed in these cells, and is located within vesicular structures in the cytosol and not on the membrane. Taken together the data demonstrate that FcRn is expressed in functionally active form in endothelial cells, indicating that these cells are a possible site at which serum IgG homeostasis is maintained.

Increasing the serum persistence of an IgG fragment by random mutagenesis.

The major histocompatibility complex (MHC) class I-related receptor FcRn is involved in regulating serum gammaglobulin (IgG) levels in mice. With the aim of increasing the serum half-life of a recombinant murine Fc gamma 1 fragment, the affinity for binding to FcRn at pH 6.0 has been increased by random mutagenesis of Thr252, Thr254, and Thr256 followed by selection using bacteriophage display. These residues were chosen as they are in proximity to the FcRn-IgG (Fc) interaction site. Two mutants with higher affinity (due to lower off-rates) than the wild-type Fc have been isolated and analyzed in pharmacokinetic studies in mice. The mutant with the highest affinity has a significantly longer serum half-life than the wild type fragment, despite its lower off-rate from FcRn at pH 7.4. The results provide support for the involvement of FcRn in the homeostasis of serum IgGs in mice. The indications that a homologous FcRn regulates IgG levels in humans suggest that this approach has implications for increasing the serum persistence of therapeutic antibodies.

The use of cyclosporine, FK506, and SDZ NIM811 to prevent CD25- quiescent peripheral blood mononuclear cells from producing human immunodeficiency virus.

It has been shown that the combined use of two pharmacologic agents can inhibit human immunodeficiency virus (HIV) production by peripheral blood mononuclear cells in vitro. One, an anti-CD25 immunotoxin (IT), kills activated T cells that produce virus; the other, the immunosuppressive drug cyclosporine, prevents the quiescent cells, which harbor HIV, from becoming activated. The present study compares the antiviral activities of two agents, SDZ NIM811 and FK506, to that of cyclosporine. In combination with the anti-CD25 IT, these drugs significantly suppressed virus production. In the absence of prior addition of the IT, the ability of the drugs to inhibit virus production was much lower, suggesting that they work effectively in latently infected cells. In the case of SDZ NIM811, the inhibition of virus production was accompanied by a modest inhibition of cell proliferation. In contrast, FK506 exerted strong antiproliferative activity. Cyclosporine was both moderately antiproliferative and a potent antiviral agent.

T cell activation and human immunodeficiency virus replication after influenza immunization of infected children.

BACKGROUND: T cell activation plays a major role in the ability of HIV to remain latent or to establish a productive infection. It has been hypothesized that vaccination-mediated immune stimulation can activate T cells and enhance HIV replication. Our study was designed to determine whether influenza immunization would induce T cell activation and increase HIV burdens in HIV-infected children. METHODS: Blood samples from 16 HIV-infected children ages 6 months to 14 years were obtained immediately before and 2 and 6 to 8 weeks after the administration of influenza vaccine. The percentage of activated (CD25+) T cells was determined by flow cytometry, and HIV viral load was measured by quantitative cultures of peripheral blood mononuclear cells and plasma HIV RNA. RESULTS: The administration of influenza vaccine was associated with significant increases in HIV viral load in 5 of 16 children evaluated. These increases in HIV burden were transient, and in four of five patients the plasma HIV RNA copy number returned to baseline 6 to 8 weeks after immunization. There was no correlation between the patient's immunologic or clinical category according to the CDC classification and either the initial viral load or the likelihood of having a significant increase after immunization. Four of the five patients who experienced increases in viral load after influenza immunization were not receiving antiretroviral therapy. CONCLUSIONS: Our results emphasize the need for additional studies that examine the effect of routine immunizations on T cell activation and HIV replication in HIV-infected children.

Expression of CD25 defines peripheral blood mononuclear cells with productive versus latent HIV infection.

The present studies were designed to further determine whether the CD25 marker could distinguish between cells productively and latently infected with HIV. This was accomplished by combining immunotoxin (IT)-mediated killing of CD25+ cells, highly sensitive indirect immunofluorescence to detect remaining CD25+ cells, and PCR-mediated amplification of proviral DNA in immunotoxin-treated vs untreated HIV-infected cells. Our results demonstrate that: 1) By direct immunofluorescence 3 to 8% of PBMCs are CD25+, whereas by indirect immunofluorescence 30% are CD25+. The increased number of CD25+ cells is due to their detection by the highly sensitive indirect immunofluorescence assay. Up to 60% of the CD25+ cells are CD4+ and 12% are CD8+. 2) Treatment of HIV-infected PBMCs with an anti-CD25 IT for 6 days eliminated both CD25high and CD25low cells and decreased the production of p24 by 99%. 3) Differences in the HIV proviral genome were detected in the unfractionated PBMCs vs PBMCs from which CD25+ cells had been eliminated by IT treatment. Hence, PBMCs containing both CD25+ and CD25- cells express all intermediate proviral species and full-length double-stranded proviral DNA. In contrast, CD25- quiescent cells contain predominantly intermediate species. These results confirm and extend our previous observations that expression of CD25 can distinguish latently infected cells from cells producing virus.

HPLC-monitoring of AZT in HIV-infected patient's plasma: a critical study.

3'-azido-2', 3'-deoxythymidine (AZT) concentrations in spiked human plasma were determined by means of reversed-phase high performance liquid chromatography (RP-HPLC). Samples were first cleaned-up for analysis using solid-phase extraction (SPE) columns filled with Silipore C18. In the concentration range comprising usual peak plasma concentrations during AZT therapy (0.1-20 mumol/l, i.e. 0.026-5.34 micrograms/ml) mean efficiency of the extraction procedure reached as high as 75.3% of original AZT concentrations in standard unextracted aqueous solutions. Replicate analyses in this range gave satisfactory intra-assay precision and reproducibility with coefficient of variation less than 11.3%. Calibration curves both in water and plasma showed good linearity (r > 0.999). The detection limit in plasma was 2 mumol/l, i.e. 5.3 ng per a 20 microliter of sample injected to the HPLC column. Plasma levels of AZT after a single dose administration, determined by HPLC and RIA showed rather poor correlation (r = 0.8900). In RIA about 1.7-4.5 times higher concentration values were obtained in a relatively short time, and, consequently, this method may better fulfil the needs of routine drug monitoring.

Amino acid analysis of selected reversed-phase high performance liquid chromatographic peaks of crude and partially purified lysed human leukocyte ultrafiltrate.

Analytic reversed-phase high performance liquid chromatography (RP-HPLC) was performed to separate from the crude lysed human leukocyte ultrafiltrate (LLU) its partially purified most immunoactive subfraction P2/II in vivo. Under conditions used, the highest degree of segregation of both. LLU and P2/II could be observed in the first, as well as in the last two fifth of the water-methanol gradient. The comparison of the RP-HPLC traces of LLU and P2/II suggests that probably some hydrophilic components of LLU have been removed or--at least--diminished. The preliminary amino acid analysis (AA) of the selected peaks showed that none of them lacks Gly, Ser, and Glu. Of the basic amino acid residues Lys has been found with relatively many peaks while hydrophobic as well as aromatic amino acids have been represented very modestly. Further study is warranted in order to determine better the bearings of presented findings for the in vivo situation.

Semipurified human leukocyte ultrafiltrate in herpes zoster. I. Large-scale preparation and biochemical analysis.

Nine batches of lysed human leukocyte ultrafiltrate (LLU) prepared from buffy coats of random healthy donors, as well as their semipurified subfractions--P2/II--were compared in terms of protein, orcinol-reactive material (ORM) content, and ratios of the average values of their ORM and protein contents. Two-step ethanol precipitation and size exclusion chromatography on Sephadex G-15 were used for partial purification and concentration. In comparison to the starting material, approximately 4.4 - fold increase in the ORM/protein ratio of P2/II has been effected. Relatively high variation in both, protein and ORM content of the crude LLU individual batches (ranges: 365 micrograms/1 ml - 962 micrograms/1 ml; 157 micrograms/1 ml - 660 micrograms/1 ml, respectively), as well as of those of P2/II fractions (ranges: 16.5 micrograms/1 ml - 207.5 micrograms/1 ml; 150 micrograms/1 ml - 480 micrograms/1 ml, respectively) could be observed. The suggested combined purification procedure removed from the LLU about 85% proteins and 33% ORM. The removed material contained inhibitors of the cell-mediated immunity (CMI)-inducing and/or augmenting properties of LLU. This is in good agreement with the observed improved therapeutic effect of P2/II fraction in herpes zoster treatment of otherwise noncompromised adults, as described in the companion paper.

UV-absorbance profile of human leukocytic ultrafiltrate after affinity chromatography on immobilized m-aminophenyl boronic acid: implication for transfer factor purification.

The UV-absorbance of the lysed human leukocyte ultrafiltrate (LLU) was investigated by Sephadex G-15 gel permeation chromatography before and after affinity chromatography on immobilized m-aminophenyl boronic acid. This biospecific fractionation caused substantial changes in 260/280-nm absorbance ratio of individual peaks as revealed by Sephadex G-15 gel permeation chromatography. While the material which had passed through the affinity support unretarded (W) appeared to be partly or completely deprived of peak IV, peaks IV and V were fairly enriched in the retarded material (R). This material, on the other hand, nearly completely missed peaks III, VI and VII. Peak III was copiously represented in material W. Boronate affinity chromatography caused enrichment in the protein content as well as in the content of purine/pyrimidine-bound ribose of material R, i.e. cis-diol group containing (RNA-like) material, considered to represent functional transfer factor activity. The described findings further support the use of immobilized derivatives of boronic acid in the separation of coplanar cis-diol group containing compounds from other constituents of LLU.

Amino acid sequence of the N-terminal region of human hemopexin.

Cyanogen bromide digestion of hemopexin at its 6 methionine residues results in 7 fragments (CB1-CB7) partially connected by disulfide bridges. By sequence studies of fragments CB1-CB4 and peptides prepared by their enzyme cleavage, a continuous amino acid sequence of the N-terminal region of human hemopexin, comprising 220 amino acid residues, was determined. The presence of intramolecular disulfide bonds, connecting half-cystine residues 126/130 and 165/170, was proved in fragments CB2 and CB3. Fragments CB1-CB4 include 5 sites, where hexosamine oligosaccharides are attached (positions 1,41,164,217 and probably 223). In the sequenced region two sites sensitive to acid hydrolysis--bonds ... Asp--Pro ... in positions 20/21 and 187/188 were found. In spite of the fact that pooled material of many donors was studied, no sequence heterogeneity was discovered.

Isolation of human haemopexin by bioaffinity chromatography on haeme-sepharose.

A preparative procedure was developed for the isolation of human apohaemopexin from Cohn fraction IV or blood serum, based on bioaffinity chromatography on haeme-Sepharose. The isolation is carried out in the pH range 4-8; hence the possibility of degradation of the carbohydrate moiety of the glycoprotein in the acidic media used in other isolation procedures is decreased. Owing to the conditions of the separation and the good stability of the affinity support, the column can be used repeatedly for long periods without a significant loss of binding capacity. The reversibility of the conformational changes that haemopexin undergoes in acidic media was examined by hydrophobic chromatography. The original hydrophobic characteristics were restored only approximately 48 h after haemopexin had been brought into a neutral medium.