Glioblastoma patients are immunosuppressed, yet glioblastomas are highly infiltrated by monocytes/macrophages. Myeloid-derived suppressor cells (MDSC; immunosuppressive myeloid cells including monocytes) have been identified in other cancers and correlate with tumor burden. We hypothesized that glioblastoma exposure causes normal monocytes to assume an MDSC-like phenotype and that MDSC are increased in glioblastoma patients. Healthy donor human CD14(+) monocytes were cultured with human glioblastoma cell lines. Controls were cultured alone or with normal human astrocytes. After 48 hours, glioblastoma-conditioned monocytes (GCM) were purified using magnetic beads. GCM cytokine and costimulatory molecular expression, phagocytic ability, and ability to induce apoptosis in activated lymphocytes were assessed. The frequency of MDSC was assessed by flow cytometry in glioma patients' blood and in GCM in vitro. As predicted, GCM have immunosuppressive, MDSC-like features, including reduced CD14 (but not CD11b) expression, increased immunosuppressive interleukin-10, transforming growth factor-beta, and B7-H1 expression, decreased phagocytic ability, and increased ability to induce apoptosis in activated lymphocytes. Direct contact between monocytes and glioblastoma cells is necessary for complete induction of these effects. In keeping with our hypothesis, glioblastoma patients have increased circulating MDSC compared with normal donors and MDSC are increased in glioma-conditioned monocytes in vitro. To our knowledge, this has not been reported previously. Although further study is needed to directly characterize their origin and function in glioblastoma patients, these results suggest that MDSC may be an important contributor to systemic immunosuppression and can be modeled in vitro by GCM.

Transferrin receptors detected by a solid-phase binding assay were shown to be specific for the host's transferrin in the representative bacterial pathogens Neisseria meningitidis (human), Pasteurella haemolytica (bovine), and Actinobacillus pleuropneumoniae (porcine). Consistent with the receptor specificity, iron-deficient bacteria were only capable of utilizing transferrin from the host as a source of iron for growth.

The nucleoside adenosine has been shown to control the production of proinflammatory molecules through its actions on cell surface purine receptors. Previously, we have reported that the adenosine A1 receptor (A1AR) regulates tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) expression and exhibits diminished function in patients with multiple sclerosis (MS; Mayne et al., Ann Neurol 1999;45:633-639). In the present study, A1AR expression in both brain and peripheral blood mononuclear cells (PBMC) from MS and control groups was characterized by fluorescence-activated cell sorting (FACS), reverse transcriptase-polymerase chain reaction (RT-PCR), and immunohistochemical analyses. FACS analyses of PBMC revealed that A1AR expression was chiefly detectable on CD14-positive cells and was reduced by 53.1% (p < 0.01) in MS patients compared to controls. A1AR mRNA levels were reduced by 43.1% (p < 0.001) in the brains of MS patients compared to patients with other neurological diseases and controls. A1AR protein expression in brain was detected primarily in CD45-positive glial cells and was markedly diminished in MS patients. The analysis of A1AR transcripts in the brain revealed that the A1AR-beta transcript was diminished (49.2%) in MS patients compared to controls (p < 0.002). These results indicate that the A1AR, expressed principally on cells of monocyte/macrophage lineage in both brain and blood, is selectively diminished in MS patients. Reduction of the A1AR-beta transcript in MS patients suggests that dysregulated splicing may influence A1AR protein levels, potentially leading to increased macrophage activation and central nervous system inflammation.

All six strains of Actinobacillus pleuropneumoniae screened for the ability to use different transferrins as a source of iron for growth were capable of using porcine but not human, bovine, or avian transferrins. A specific binding activity for porcine transferrin (pTf) was expressed in cells grown in the presence of specific iron-chelators and was repressed by addition of excess iron. Two iron-repressible outer-membrane proteins of 105 and 56 kD were specifically isolated from serotype 1, 2 and 7 strains of A. pleuropneumoniae by an affinity-isolation method using biotinylated porcine transferrin and streptavidin-agarose.

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This study was done primarily to determine whether the previously observed specificity of the meningococcal transferrin and lactoferrin receptors for human proteins was maintained in vivo during meningococcal infection in mice. Preliminary experiments evaluating the choice of host strain, the age and sex of mice, and the growth conditions of the meningococci indicated that 45-day-old female Swiss Webster mice challenged with meningococci grown on low-pH, low-iron Mueller-Hinton agar plates were appropriate for this study. The comparison of transferrins and lactoferrins from different species demonstrated that only the human forms of these proteins were utilized by meningococci; there was significantly greater mortality among mice treated with iron-saturated human transferrin or lactoferrin (93 and 100%, respectively) than among those not treated or treated with iron-saturated bovine transferrin or bovine lactoferrin (0%). Provision of exogenous hemoglobin also resulted in increased mortality, although not as great as that observed with amounts of transferrin with equivalent iron content, which parallels the more effective utilization of transferrin and lactoferrin in in vitro growth experiments. In addition, unlike with transferrin and lactoferrin, there was no difference in utilization of human and bovine hemoglobin in vitro or in vivo.

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The tbpA and *pi? genes encoding the transferrin receptor proteins T b p l and Tbp2 from a serotype 7 strain of Actinobacillus pleuropneumoniae were cloned, sequenced, and expressed in Escherichia coli. The tbpB gene was preceded by putative promoter and regulatory sequences and was separated from the downstream tbpA gene by a 13 bp intercistronic sequence suggesting that the two genes may be coordinately transcribed. Determination of the nucleotide sequence of this region facilitated PCR amplification of the tbp region from a serotype 1 strain for comparative purposes. The deduced amino acid sequences of the T b p l proteins had regions of homology with Neisseria Lbp and Tbpls and with TonB-dependent outer membrane (OM) receptors of E. coli. The deduced amino acid sequences of the Tbp2 proteins were nearly identical to those presented in previous studies. Upon high-level expression of the tbpA gene, a large proportion of the recombinant T b p l was found in inclusion bodies and could not be aff inity-isolated with immobilized porcine transferrin. Most of the remaining expressed T b p l was present in the OM fraction, was expressed at the surface of E. coli cells, and retained binding activity that was specific for the Globe of porcine transferrin. Although recombinant Tbp2 was found in inclusion bodies during high-level expression, a significant proportion was associated with a novel OM fraction that appeared in sucrose density gradients which was distinct from the OM fraction containing recombinant Tbpl. The recombinant Tbp2 was accessible at the surface yet was unable to bind porcine transferrin. In contrast to previous observations, the binding by recombinant Tbp2 was specific for the Globe of porcine transferrin. These results indicate that the A. pleuropneumoniae transferrin receptor proteins have similar properties to the receptor proteins in Neisseria spp. and Haemophilus influenzae, and that functional studies performed with recombinant receptor proteins need to consider differences in processing and export of these proteins when expressed in heterologous hosts.