Biochemical and immunological properties of cytokines conjugated to dendritic polymers.

Here we describe a post-translational modification of SC-63032, a variant of the species restricted, multi-lineage hematopoeitic factor human interleukin-3 (hIL-3). We have made two new dendritic polymer (polyamidoamine or PAMAM dendrimers, generation 5)-SC-63032 bioconjugates. Using two distinct chemistries (one of which is novel to this work), we achieved site-specific conjugation with respect to the amino acid in the proteins ligated to the dendrimers. In both bioconjugates, conjugated cytokine maintains its ability to bind the hIL-3 alpha receptor subunit, but is significantly (about 10-fold) less potent in inducing hIL-3 dependent in vitro cell proliferation than is the free cytokine. In vivo data indicates that conjugation decreases the immunogenicity of the conjugated cytokine modestly. In the absence of pharmacokinetic or biodistribution effects associated with the bioconjugates that increase their potency in vivo (which can only be tested in a higher primate, due to the species restriction of hIL-3 and its derivatives), these immune mitigation effects may be too small to be therapeutically significant. Though unmodified PAMAM dendrimers fail to elicit an antibody response in mice, protein conjugation to dendrimers haptenizes them, and a dendrimer-specific antibody response is produced. In toto, the principal limitation of the dendrimer-cytokine bioconjugates herein is in their reduced receptor affinity and potency in vitro. Were the in vivo potency of the bioconjugates to parallel the in vitro potency of the conjugates reported here, it is likely that particular dendrimer bioconjugates could not justify their higher costs of goods relative to the parent SC-63032 molecule, though retention of SC-63032 biological activities in conjugates suggests that other cytokine-dendrimer bioconjugates may be bioactive. This is good news to the nanotechnology community, in as much as PAMAM dendrimers are among the monodisperse polymeric nanomaterials available, and these results show that they can be used successfully in conjugates to bioactive proteins.

The enhanced in vitro hematopoietic activity of leridistim, a chimeric dual G-CSF and IL-3 receptor agonist.

The in vitro activity of leridistim was characterized for cell proliferation, generation of colony-forming units (CFU) and differentiation of CD34+ cells. In AML-193.1.3 cells, leridistim exhibited a significant increase in potency compared to rhG-CSF, SC-65303 (an IL-3 receptor agonist) or an equimolar combination of rhG-CSF and SC-65303. CFU-GM assays demonstrated that at 50% of the maximum response, the relative potency of leridistim was 12-fold greater than the combination of rhG-CSF and rhIL-3 and 44-fold more potent than rhG-CSF alone. In multi-lineage CFU assays, a combination of erythropoietin (rhEPO) and leridistim resulted in greater numbers of BFU-E, CFU-GEMM and CFU-Mk than rhEPO alone. Ex vivo culture of peripheral blood or bone marrow CD34+ cells with leridistim substantially increased total viable cells over cultures stimulated with rhG-CSF, SC-65303, or a combination of rhG-CSF and SC-65303. Culture with leridistim, resulted in a greater increase in myeloid (CD15+/CD11b+), monocytic (CD41-/CD14+) and megakaryocytic (CD41+/CD14-) precursor cells without depleting the progenitor pool (CD34+/CD15-/CD11b-). These results demonstrate that leridistim is a more potent stimulator of hematopoietic proliferation and differentiation than the single receptor agonists (rhG-CSF and SC-65303) either alone or combined. These unique attributes suggest that leridistim may enhance hematopoietic reconstitution following myelosuppressive chemotherapy.

Promegapoietin, a family of chimeric growth factors, supports megakaryocyte development through activation of IL-3 and c-Mpl ligand signaling pathways.

OBJECTIVE: The signaling pathways induced by promegapoietin (PMP), a family of chimeric growth factors that activate the human IL-3 and c-Mpl receptors, were investigated. METHODS: The biological activity of PMP was examined by receptor binding, cell proliferation, ex vivo expansion of hematopoietic progenitor cells, and in vivo production of platelets. The activation of signaling pathways was examined by Western blot and Northern blot analyses. RESULTS: Two PMP molecules, PMP-1 and PMP-1a, induced proliferation of cells expressing the IL-3 receptor, c-Mpl, or both receptors and bound to the IL-3 receptor and c-Mpl with high affinity. Ex vivo expansion assays using human bone marrow CD34(+) cells suggested that PMP-1 induced greater total cellular expansion as well as expansion of CD41(+) megakaryocytic precursor cells than IL-3 or c-Mpl ligand alone. Subcutaneous administration of 50 microg/kg of PMP-1 for 10 days to rhesus monkeys resulted in increased platelet production in vivo from a baseline of 357 +/- 45 x 10(3) cells/mL to 1376 +/- 151 x 10(3) cell/mL. PMP-1 induced phosphorylation of the beta(c) subunit of IL-3 receptor and c-Mpl, JAK2, and STAT5b, but not STAT3. PMP-1 induced greater expression of Pim-1, c-Myc, and cyclin D2 than did either an IL-3 receptor agonist or c-Mpl receptor agonist alone. The magnitude of induction of early response genes was similar for PMP and the coaddition of IL-3 receptor agonist and c-Mpl receptor agonist. CONCLUSION: PMP combines the biological activities of IL-3 and c-Mpl ligand in a single molecule that can simultaneously activate signaling pathways induced by both these ligands.

Phage display mutagenesis of the chimeric dual cytokine receptor agonist myelopoietin.

Myelopoietins comprise a class of chimeric cytokine receptor agonists consisting of an hIL-3 (human interleukin-3) receptor agonist and an hG-CSF (human granulocyte colony-stimulating factor) receptor agonist linked head-to-tail at their respective carboxy and amino termini. The combination of an early acting cytokine (hIL-3) with a late acting one (hG-CSF) allows efficient hematopoeitic reconstruction following myeloablative insult, and drives differentiation of non-myelocytic lineages (ie thrombocytic lineages) that are inaccessible using hG-CSF alone, in both preclinical models and clinical settings. A myelopoietin species was displayed and mutagenized on filamentous bacteriophage: both component agonists of myelopoietin were presented in biologically functional conformations as each recognized its corresponding receptor. Five amino acid positions in a short region of the hG-CSF receptor agonist module of myelopoietin that had been identified as important for proliferative activity were mutagenized. Display was used because it allows very 'deep' mutagenesis at selected residues: >10(5) substitution variants were affinity-screened using the hG-CSF receptor and 130 new, active variants of myelopoietin were identified and characterized. None of the selected variants were significantly more active than the parental myelopoietin species in a hG-CSF-dependent cell proliferation assay, though many were as active. Many of these relatively high-activity variants contained parental amino acids at several positions, suggesting the parental sequence may already be optimal at these positions for the assays used, and potentially accounting for the failure to identify enhanced bioactivity variants. Analysis of substitutions of high-activity variants complements and extends previous alanine scanning, and other genetic and biochemical data for hG-CSF variants.

Circular permutation of granulocyte colony-stimulating factor.

The sequence of granulocyte colony-stimulating factor (G-CSF) has been circularly permuted by introducing new chain termini into interhelical loops and by constraining the N- and C-terminal helices, either by direct linkage of the termini (L0) or by substitution of the amino-terminal 10-residue segment with a seven-residue linker composed of glycines and serines (L1). All the circularly permuted G-CSFs (cpG-CSFs) were able to fold into biologically active structures that could recognize the G-CSF receptor. CD and NMR spectroscopy demonstrated that all of the cpG-CSFs adopted a fold similar to that of the native molecule, except for one [cpG-CSF(L1)[142/141]] which has the new termini at the end of loop 34 with the shorter L1 linker. All of the cpG-CSFs underwent cooperative unfolding by urea, and a systematically lower free energy change (DeltaGurea) was observed for molecules with the shorter L1 linker than for those molecules in which the original termini were directly linked (the L0 linker). The thermodynamic stability of the cpG-CSFs toward urea was found to correlate with their relative ability to stimulate proliferation of G-CSF responsive cells. Taken together, these results indicate that the G-CSF sequence is robust in its ability to undergo linear rearrangement and adopt a biologically active conformation. The choice of linker, with its effect on stability, seems to be important for realizing the full biological activity of the three-dimensional structure. The breakpoint and linker together are the ultimate determinants of the structural and biological profiles of these circularly permuted cytokines. In the following paper [McWherter, C. A., et al. (1999) Biochemistry 38, 4564-4571], McWherter and co-workers have used circularly permuted G-CSF sequences to engineer chimeric dual IL-3 and G-CSF receptor agonists in which the relative spatial orientation of the receptor agonist domains is varied. Interpreting the differences in activity for the chimeric molecules in terms of the connectivity between domains depends critically on the results reported here for the isolated cpG-CSF domains.

Circular permutation of the granulocyte colony-stimulating factor receptor agonist domain of myelopoietin.

Myelopoietins (MPOs) are a family of engineered dual interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF) receptor agonists that are superior in comparison to the single agonists in their ability to promote the growth and maturation of hematopoietic cells of the myeloid lineage. A series of MPO molecules were created which incorporated circularly permuted G-CSF (cpG-CSF) sequences with an IL-3 receptor (IL-3R) agonist moiety attached at locations that correspond to the loops that connect the helices of the G-CSF four-helix bundle structure. The cpG-CSF linkage sites (using the original sequence numbering) were residue 39, which is at the beginning of the first loop connecting helices 1 and 2; residue 97, which is in the turn connecting helices 2 and 3; and residues 126, 133, and 142, which are at the beginning, middle, and end, respectively, of the loop connecting helices 3 and 4. The N- and C-terminal helices of each cpG-CSF domain were constrained, either by direct linkage of the termini (L0) or by replacement of the amino-terminal 10-residue segment with a seven-residue linker composed of SGGSGGS (L1). All of the MPO molecules stimulated the proliferation of both IL-3-dependent (EC50 = 13-95 pM) and G-CSF-dependent (EC50 = 35-710 pM) cell lines. MPOs with the IL-3R agonist domain linked to cpG-CSFs in the first (residue 39) or second (residue 133) long overhand loops were found by CD spectroscopy to have helical contents similar to that expected for a protein comprised of two linked four-helix bundles. The MPOs retained the ability to bind to the IL-3R with affinities similar to that of the parental MPO. Using both a cell surface competitive binding assay and surface plasmon resonance detection of binding kinetics, the MPOs were found to bind to the G-CSF receptor with low nanomolar affinities, similar to that of G-CSF(S17). In a study of isolated cpG-CSF domains [Feng, Y., et al. (1999) Biochemistry 38, 4553-4563], domains with the L1 linker had lower G-CSF receptor-mediated proliferative activities and conformational stabilities than those which had the L0 linker. A similar trend was found for the MPOs in which the G-CSFR agonist activity is mostly a property of the cpG-CSF domain. Important exceptions were found in which the linkage to the IL-3R agonist domain either restored (e.g., attachment at residue 142) or further decreased (linkage at residue 39) the G-CSFR-mediated proliferative activity. MPO in which the IL-3R agonist domain is attached to the cpG-CSF(L1)[133/132] domain was shown to be more potent than the coaddition of the IL-3R agonist and G-CSF in stimulating the production of CFU-GM colonies in a human bone marrow-derived CD34+ colony-forming unit assay. Several MPOs also had decreased proinflammatory activity in a leukotriene C4 release assay using N-formyl-Met-Leu-Phe-primed human monocytes. It was found that circular permutation of the G-CSF domain can alter the ratio of G-CSFR:IL-3R agonist activities, demonstrating that it is a useful tool in engineering chimeric proteins with therapeutic potential.

Phage presentation and affinity selection of a deletion mutant of human interleukin-3.

A deletion derivative of the cytokine human interleukin-3 (hIL-3(15-125), comprising amino acids 15-125 of the native protein) was produced as a fusion to the filamentous phage surface protein pIII. The cytokine was detected in association with phage particles by protein immunoblotting. Compared to an equivalent quantity of soluble-cytokine, phage-presented hIL-3(15-125) exhibited reduced biological activity in a hIL-3-dependent cell proliferation assay. The reduction in activity was attributable to presence of phage particles in the assay, rather than directly owing to physical incorporation of the cytokine into the phage particle. Owing to the position of the amber codon in the phagemid vector, the phagemid-produced free hIL-3(15-125) species (designated hIL-3(15-125) epsilon) had 20 amino acids appended to its C-terminus; hIL-3(15-125) epsilon did not exhibit reduced bioactivity. hIL-3(15-125)-presenting phage were affinity-selected with either a hIL-3-reactive polyclonal antibody or with cells expressing the heterodimeric hIL-3 receptor. These data are consistent with the use of phage-display technology for the affinity selection of hIL-3 variants with modified biological properties.

Multiple conformations of a human interleukin-3 variant.

Interleukin-3 (IL-3) is a cytokine that stimulates the proliferation and differentiation of hematopoietic cells. The hyperactive hIL-3 variant SC-55494 was shown to have at least two major conformations by high-resolution NMR spectroscopy. Mutants of SC-55494 were constructed in which alanine was substituted for proline in order to test the hypothesis that proline cis-trans isomerization is the source of the observed conformational heterogeneity, as well as to evaluate the effect of prolyl peptide bond configuration on biological activity. NMR spectra of four single proline-to-alamine mutants (P30A, P31A, P33A, and P37A) retain doubled resonances, while spectra of the double mutant P30A/P31A and the quadruple mutant P30A/P31A/P33A/ P37A are substantially free of heterogeneity. These observations suggest that the two major conformations in SC-55494 correspond to cis and trans isomers of either or both of the R29-P30 and P30-P31 peptide bonds. All six mutants had somewhat lower cell proliferative activity than SC-55494, with relative activities ranging from 40 to 80%. The P37A mutant has a binding affinity to the low-affinity IL-3 receptor alpha-subunit statistically equivalent to SC-55494, while P30A, P31A, and P33A each had about two-fold decreases, and P30A/P31A and P30A/P31A/P33A/P37A had four-fold decreases. These findings suggest an important role for the cis configuration of either or both of the R29-P30 and P30-P31 peptide bonds in IL-3 for optimal interaction with the receptor alpha-subunit.

[Phototherapy of behavioral disorders as a sequela of disordered circadian rhythm in dementia in the elderly--difficulties in practical clinical applications]. / Lichttherapie von Verhaltensstörungen als Folge gestörter zirkadianer Rhythmen bei dementiellen Alterspatienten--Schwierigkeiten der praktisch-klinischen Anwendbarkeit.

The behavioral disturbances of 16 nursing home patients with a history of sundowning behaviour and dementia were treated with light therapy instead of psychopharmacological medication. After the withdrawal of the before established medication a very individual, heterogenous deterioration of the symptoms was noted. A significant improvement of the behaviour was not found by light therapy in any patient. The problems in practical handling and possible consequences of further investigation are discussed.

Amphotericin B selectively stimulates macrophages from high responder mouse strains.

Lymphoid cells from most inbred mouse strains respond to amphotericin B (AmB)-induced immunostimulation. However, C57BL/6 mice and related strains display low or absent lymphoid cell stimulation by AmB and enhanced susceptibility to AmB toxicity. Experiments reported here show that in vitro incubation with AmB can stimulate AKR (AmB-high responder strain) macrophage proliferation. Intraperitoneal injection of AKR mice with AmB also elicits a population of macrophages primed for enhanced oxidative burst activity after triggering by zymosan particles. Under the same experimental conditions, AmB elicits a population of very weakly responsive macrophages from C57BL/6 mice. The low responsiveness of C57BL/6 macrophages correlates with previous observations that AmB is a potent immunoadjuvant and B cell mitogen in most inbred strains, but it selectively lacks immunoadjuvant effects in C57BL/6 mice and it also fails to induce polyclonal B cell stimulation in their spleen cell suspensions. Similarly, in measurements of protein synthesis in vitro, high concentrations of AmB produce a greater inhibition of protein synthesis in C57BL/6 peritoneal macrophages than in parallel cultures of AKR macrophages. These findings support the hypothesis that the macrophage is an important target cell in the mediation of AmB-induced immunomodulation.

Effects of Histoplasma capsulatum on murine macrophage functions: inhibition of macrophage priming, oxidative burst, and antifungal activities.

Histoplasma capsulatum yeast cells fail to trigger an oxidative burst response in normal murine macrophages. The results of this study, in which an in vitro assay of macrophage antifungal effects was used, extend these findings. During 18 h of incubation, unprimed elicited murine macrophages inhibited H. capsulatum growth only when macrophages were present in great excess. Gamma interferon (IFN-gamma)-primed macrophages showed enhanced fungal growth inhibition but a similar requirement for an excess of phagocytes. Macrophages containing heat-killed H. capsulatum exhibited diminished antifungal effects toward viable H. capsulatum and Saccharomyces cerevisiae cells. Parallel experiments showed no comparable effect of ingested latex particles on macrophage antifungal activity. Using chemiluminescence as a measure of the oxidative burst, we found that macrophages primed in vitro with IFN-gamma alone failed to exhibit a significant response to triggering by H. capsulatum yeast cells unless a second priming agent (tumor necrosis factor alpha or bacterial lipopolysaccharide) was added to IFN-gamma. Furthermore, macrophage priming with single agents was blocked by the prior ingestion of heat-killed H. capsulatum. These studies provide evidence that ingestion of H. capsulatum yeast cells can induce a prompt and enduring deactivation of murine macrophages.

The relationship between adjuvant and mitogenic effects of amphotericin methyl ester.

The antifungal polyene amphotericin B (AmB) and its methyl ester derivative (AME) both show potent murine immunostimulant as well as B-cell activating effects. Under certain experimental conditions, AME is a much more potent polyclonal B-cell activator (PBA) than AmB. Notable features of the murine B-cell stimulation induced by AME include: (i) High concentrations of AME (50-100 microgram/ml) are required and even at this level exhibit little or no spleen cell toxicity. (ii) Several lines of evidence suggest that the B-cell activating properties of AME are not involved in the cellular mechanism of adjuvant activity in vivo. (iii) There is a strong correlation between the magnitude of the in vitro PBA effects and the in vivo adjuvant effects of AME in a survey of different mouse strains. This evidence suggests that there is genetic control of the murine lymphoid cell-stimulatory effects of AME and that a small number of genes determines the responsive phenotype.

Effect of exogenous lipids and lipoproteins on the primary immune response in vitro.

Cholesterol and certain lipoproteins have regulatory effects on the primary immune responses of murine spleen cells in vitro. The plaque-forming cell (PFC) responses to sheep red blood cells of trinitrophenylated Brucella abortus were studied in complete, lipid-depleted or lipoprotein-reconstituted media. The requirement for exogenous low density lipoprotein (LDL) and its cholesterol moiety was established by comparison of the yield of PFC in cell cultures deprived of lipoproteins with that in cultures to which specific classes of lipoproteins were added. The spleen cells in complete medium yielded about 10-fold greater PFC responses than cells in lipoprotein-deficient medium. In lipoprotein-deficient media, human LDL completely reversed the decreased immune response, LDL lipids and free cholesterol partially reversed the deficit, the human high density lipoproteins and an apo B phospholipid complex were ineffective. In complete media, cholesterol at higher concentrations (100--200 microgram/ml) and LDL lipids partially inhibited the primary immune response. Exogenous cholesterol was required for the in vitro response to both thymus-dependent and thymus-independent antigens.

[Antibodies against native deoxyribonucleic acid (anti-nDNA) without antinuclear antibodies: clinical significance (author's transl)]. / Antikörper gegen native Desoxyribonucleinsäure ohne antinukleäre Antikörper.

Antinuclear antibodies (ANA) by indirect immunofluorescence and antibodies against native deoxyribonucleic acid (anti-nDNA) by radiommunoassay were measured simultaneously in 6,000 sera from about 5,000 patients. Usual findings about 5,000 patients. Usual findings were: (a) both tests negative (75%), (b) only ANA positive (20%), and (c) both tests positive (3%). The unusual combination of ANA-negative/anti-nDNA positive was found in 117 sera from 24 patients. These patients were examined more closely clinically and the mentioned tests repeated. In five with systemic lupus erythematosus, in two with chronic rheumatoid arthritis and one with chronic urticaria this finding occurred repeatedly over some time. In five additional patients (two with chronic hepatitis, one each with drug-induced systemic lupus erythematosus, chronic rheumatoid arthritis, and drug related haemolytic anaemia) this unusual finding occurred only once but with high levels of anti-nDNA. In 11 patients with various diseases the combination of ANA-negative/anti-nDNA positive occurred only once, with the anti-nDNA value being low. In a control group of patients with mononucleosis, cytomegalic disease, acute or chronic hepatitis or hepatoma, anti-nDNA results were never positive.

An immunological basis of schizophrenia and affective disorders?

In 48 patients with schizophrenia and 32 patients with affective disorders, different immune parameters were tested. Compared to blood-donors, IgG and IgM serum concentrations were increased in both the schizophrenic and affective disorders. However, these abnormalities did not differ from hospital control populations. The patients failed to show an association of antibodies considered to be characteristic of autoimmune diseases. In addition, no increased incidence of circulating immune compexes was detected. The only substantial serologic abnormality froun was an elevation of C4 levels in patients with biopolar psychosis.