Malaria transmission-blocking vaccines Pfs230D1-EPA and Pfs25-EPA in Alhydrogel in healthy Malian adults; a phase 1, randomised, controlled trial.

BACKGROUND: Malaria transmission-blocking vaccines target mosquito-stage parasites and will support elimination programmes. Gamete vaccine Pfs230D1-EPA/Alhydrogel induced superior activity to zygote vaccine Pfs25-EPA/Alhydrogel in malaria-naive US adults. Here, we compared these vaccines in malaria-experienced Malians. METHODS: We did a pilot safety study then double-blind, block-randomised, comparator-controlled main-phase trial in malaria-intense Bancoumana, Mali. 18-50-year-old healthy non-pregnant, non-breastfeeding consenting adult residents were randomly assigned (1:1:1:1) to receive four doses at months 0, 1, 4·5, and 16·5 of either 47 µg Pfs25, 40 µg Pfs230D1 or comparator (Twinrix or Menactra)-all co-administered with normal saline for blinding-or 47 µg Pfs25 plus 40 µg Pfs230D1 co-administered. We documented safety and tolerability (primary endpoint in the as-treated populations) and immunogenicity (secondary endpoint in the as-treated populations: ELISA, standard-membrane-feeding assay, and mosquito direct skin feed assay). This trial is registered at ClinicalTrials.gov, NCT02334462. FINDINGS: Between March 19, and June 2, 2015, we screened 471 individuals. Of 225 enrolled for the pilot and main cohorts, we randomly assigned 25 participants to pilot safety cohort groups of five (20%) to receive a two-dose series of Pfs25-EPA/Alhydrogel (16 µg), Pfs230D1-EPA/Alhydrogel (15 µg) or comparator, followed by Pfs25-EPA/Alhydrogel (16 µg) plus Pfs230D1-EPA/Alhydrogel (15 µg) or comparator plus saline. For the main cohort, we enrolled 200 participants between May 11 and June 2, 2015, to receive a four-dose series of 47 µg Pfs25-EPA/Alhydrogel plus saline (n=50 [25%]; Pfs25), 40 µg Pfs230D1-EPA/Alhydrogel plus saline (n=49 [25%]; Pfs230D1), 47 µg Pfs25-EPA/Alhydrogel plus 40 µg Pfs230D1-EPA/Alhydrogel (n=50 [25%]; Pfs25 plus Pfs230D1), or comparator (Twinrix or Menactra) plus saline (n=51 [25%]). Vaccinations were well tolerated in the pilot safety and main phases. Most vaccinees became seropositive after two Pfs230D1 or three Pfs25 doses; peak titres increased with each dose thereafter (Pfs230D1 geometric mean: 77·8 [95% CI 56·9-106·3], 146·4 [108·3-198·0], and 410·2 [301·6-558·0]; Pfs25 geometric mean 177·7 [130·3-242·4] and 315·7 [209·9-474·6]). Functional activity (mean peak transmission-reducing activity) appeared for Pfs230D1 (74·5% [66·6-82·5]) and Pfs25 plus Pfs230D1 (68·6% [57·3-79·8]), after the third dose and after the fourth dose (88·9% [81·7-96·2] for Pfs230D1 and 85·0% [78·4-91·5] Pfs25 plus Pfs230D1) but not for Pfs25 (58·2% [49·1-67·3] after the third dose and 58·2% [48·5-67·9] after the fourth dose). Pfs230D1 transmission-reducing activity (73·7% [64·1-83·3]) persisted 10 weeks after the fourth dose. Transmission-reducing activity of 80% was estimated at 1659 ELISA units for Pfs25, 218 for Pfs230D1, and 223 for Pfs230D1 plus Pfs25. After 3850 direct skin feed assays, 35 participants (12 Pfs25, eight Pfs230D1, five Pfs25 plus Pfs230D1, and ten comparator) had transmitted parasites at least once. The proportion of positive assays in vaccine groups (Pfs25 33 [3%] of 982 [-0·013 to 0·014], Pfs230D1 22 [2%] of 954 [-0·005 to 0·027], and combination 11 [1%] of 940 [-0·024 to 0·002]) did not differ from that of the comparator (22 [2%] of 974), nor did Pfs230D1 and combination groups differ (-0·024 to 0·001). INTERPRETATION: Pfs230D1 but not Pfs25 vaccine induces durable serum functional activity in Malian adults. Direct skin feed assays detect parasite transmission to mosquitoes but increased event rates are needed to assess vaccine effectiveness. FUNDING: Intramural Research Program of the National Institute of Allergy and Infectious Diseases and US National Institutes of Health.

Outer membrane protein complex as a carrier for malaria transmission blocking antigen Pfs230.

Malaria transmission blocking vaccines (TBV) target the mosquito stage of parasite development by passive immunization of mosquitoes feeding on a vaccinated human. Through uptake of vaccine-induced antibodies in a blood meal, mosquito infection is halted and hence transmission to another human host is blocked. Pfs230 is a gametocyte and gamete surface antigen currently under clinical evaluation as a TBV candidate. We have previously shown that chemical conjugation of poorly immunogenic TBV antigens to Exoprotein A (EPA) can enhance their immunogenicity. Here, we assessed Outer Membrane Protein Complex (OMPC), a membrane vesicle derived from Neisseria meningitidis, as a carrier for Pfs230. We prepared Pfs230-OMPC conjugates with varying levels of antigen load and examined immunogenicity in mice. Chemical conjugation of Pfs230 to OMPC enhanced immunogenicity and functional activity of the Pfs230 antigen, and OMPC conjugates achieved 2-fold to 20-fold higher antibody titers than Pfs230-EPA/AdjuPhos® at different doses. OMPC conjugates were highly immunogenic even at low doses, indicating a dose-sparing effect. EPA conjugates induced an IgG subclass profile biased towards a Th2 response, whereas OMPC conjugates induced a strong Th1-biased immune response with high levels of IgG2, which can benefit Pfs230 antibody functional activity, which depends on complement activation. OMPC is a promising carrier for Pfs230 vaccines.

Adjuvant and carrier protein-dependent T-cell priming promotes a robust antibody response against the Plasmodium falciparum Pfs25 vaccine candidate.

Humoral immune responses have the potential to maintain protective antibody levels for years due to the immunoglobulin-secreting activity of long-lived plasma cells (LLPCs). However, many subunit vaccines under development fail to generate robust LLPC responses, and therefore a variety of strategies are being employed to overcome this limitation, including conjugation to carrier proteins and/or formulation with potent adjuvants. Pfs25, an antigen expressed on malaria zygotes and ookinetes, is a leading transmission blocking vaccine (TBV) candidate for Plasmodium falciparum. Currently, the conjugate vaccine Pfs25-EPA/Alhydrogel is in Phase 1 clinical trials in the USA and Africa. Thus far, it has proven to be safe and immunogenic, but it is expected that a more potent formulation will be required to establish antibody titers that persist for several malaria transmission seasons. We sought to determine the contribution of carrier determinants and adjuvants in promoting high-titer, long-lived antibody responses against Pfs25. We found that both adjuvants and carrier proteins influence the magnitude and capacity of Pfs25-specific humoral responses to remain above a protective level. Furthermore, a liposomal adjuvant with QS21 and a TLR4 agonist (GLA-LSQ) was especially effective at inducing T follicular helper (Tfh) and LLPC responses to Pfs25 when coupled to immunogenic carrier proteins.

Impact on malaria parasite multiplication rates in infected volunteers of the protein-in-adjuvant vaccine AMA1-C1/Alhydrogel+CPG 7909.

BACKGROUND: Inhibition of parasite growth is a major objective of blood-stage malaria vaccines. The in vitro assay of parasite growth inhibitory activity (GIA) is widely used as a surrogate marker for malaria vaccine efficacy in the down-selection of candidate blood-stage vaccines. Here we report the first study to examine the relationship between in vivo Plasmodium falciparum growth rates and in vitro GIA in humans experimentally infected with blood-stage malaria. METHODS: In this phase I/IIa open-label clinical trial five healthy malaria-naive volunteers were immunised with AMA1/C1-Alhydrogel+CPG 7909, and together with three unvaccinated controls were challenged by intravenous inoculation of P. falciparum infected erythrocytes. RESULTS: A significant correlation was observed between parasite multiplication rate in 48 hours (PMR) and both vaccine-induced growth-inhibitory activity (Pearson r = -0.93 [95% CI: -1.0, -0.27] P = 0.02) and AMA1 antibody titres in the vaccine group (Pearson r = -0.93 [95% CI: -0.99, -0.25] P = 0.02). However immunisation failed to reduce overall mean PMR in the vaccine group in comparison to the controls (vaccinee 16 fold [95% CI: 12, 22], control 17 fold [CI: 0, 65] P = 0.70). Therefore no impact on pre-patent period was observed (vaccine group median 8.5 days [range 7.5-9], control group median 9 days [range 7-9]). CONCLUSIONS: Despite the first observation in human experimental malaria infection of a significant association between vaccine-induced in vitro growth inhibitory activity and in vivo parasite multiplication rate, this did not translate into any observable clinically relevant vaccine effect in this small group of volunteers. TRIAL REGISTRATION: ClinicalTrials.gov [NCT00984763].

IL-27 regulates IL-10 and IL-17 from CD4+ cells in nonhealing Leishmania major infection.

Control of infection caused by Leishmania major requires the development of IFN-gamma+CD4+ lymphocytes for the induction of microbicidal activity in host macrophages. We recently reported on the inability of conventionally resistant C57BL/6 mice to successfully resolve infection by an isolate of L. major, despite a strong IFN-gamma response by the host. Susceptibility was caused by Ag-specific IL-10 from CD4+ cells that were also producing IFN-gamma. In the present studies, we have explored the role for IL-27 in the regulation of IL-10 from Th1 cells in leishmaniasis. Cytokine analysis of CD4+ cells in the lesions and draining lymph nodes of infected IL-27R-deficient (WSX-1(-/-)) mice revealed diminished IL-10 from IFN-gamma+ CD4+ cells, which was accompanied by a reduction in total IFN-gamma+CD4+ cells and an increase in IL-4. Despite the inhibition of IL-10 from CD4+ cells, no significant change in parasite numbers was observed, due both to the shift in the Th1/Th2 balance and to residual levels of IL-10. Strikingly, infected WSX-1(-/-) mice developed more severe lesions that were associated with the appearance of IL-17+ CD4+ cells, demonstrating a function for IL-27 in blocking the development of inappropriate Th17 cells during L. major infection. The results demonstrate the pleiotropic effects that IL-27 has on L. major-driven Th1, Th2, and Th17 development, and reinforce its function as a key regulatory cytokine that controls the balance between immunity and pathology.

IL-10 and TGF-beta control the establishment of persistent and transmissible infections produced by Leishmania tropica in C57BL/6 mice.

Leishmania tropica is the causative agent of Old World anthroponotic cutaneous leishmaniasis, which is characterized by lesions that take an extended period of time to heal, often resulting in disfiguring scars, and are more refractory to treatment than leishmaniasis caused by Leishmania major. Immunologic studies involving experimental animal models of L. tropica infection are virtually nonexistent. In the current study, infectious-stage L. tropica were used to establish dermal infections in C57BL/6 and BALB/c mice. In both strains, the lesions were slow to develop and showed minimal pathology. They nonetheless contained a stable number of between 10(4) and 10(5) parasites for over 1 year, which were efficiently picked up by a natural sand fly vector, Phlebotomus sergenti. Control of parasite growth depended on the development of a Th1 response, as C57BL/6 mice genetically deficient in Th1 cytokines and BALB/c mice treated with Abs to IFN-gamma harbored significantly more parasites. By contrast, IL-10-deficient mice harbored significantly fewer parasites throughout the infection. To further study the immunologic mechanisms that may prevent efficient clearance of the parasites, IL-10 and TGF-beta signaling were abrogated during the chronic phase of infection in wild-type C57BL/6 mice. Distinct from chronic L. major infection, IL-10 blockade alone had no effect on L. tropica, but required simultaneous treatment with anti-TGF-beta Abs to promote efficient parasite clearance from the infection site. Thus, chronic infection with L. tropica appears to be established via multiple suppressive factors, which together maintain the host as a long-term reservoir of infection for vector sand flies.

Despite increased CD4+Foxp3+ cells within the infection site, BALB/c IL-4 receptor-deficient mice reveal CD4+Foxp3-negative T cells as a source of IL-10 in Leishmania major susceptibility.

BALB/c IL-4Ralpha(-/-) mice, despite the absence of IL-4/IL-13 signaling and potent Th2 responses, remain highly susceptible to Leishmania major substain LV39 due exclusively to residual levels of IL-10. To address the contribution of CD4(+)CD25(+) T regulatory (Treg) cells to IL-10-mediated susceptibility, we depleted CD4(+)CD25(+) cells in vivo and reconstituted IL-4Ralpha x RAG2 recipients with purified CD4(+)CD25(-) T cells. Although anti-CD25 mAb treatment significantly decreased parasite numbers in IL-4Ralpha(-/-) mice, treatment with anti-IL-10R mAb virtually eliminated L. major parasites in both footpad and dermal infection sites. In addition, IL-4Ralpha x RAG2 mice reconstituted with CD4(+) cells depleted of CD25(+) Treg cells remained highly susceptible to infection. Analysis of L. major-infected BALB/c and IL-4Ralpha(-/-) inflammatory sites revealed that the majority of IL-10 was secreted by the CD4(+)Foxp3(-) population, with a fraction of IL-10 coming from CD4(+)Foxp3(+) Treg cells. All T cell IFN-gamma production was also derived from the CD4(+)Foxp3(-) population. Nevertheless, the IL-4Ralpha(-/-)-infected ear dermis, but not draining lymph nodes, consistently displayed 1.5- to 2-fold greater percentages of CD4(+)CD25(+) and CD4(+)Foxp3(+) Treg cells compared with the BALB/c-infected dermis. Thus, CD4(+)Foxp3(-) T cells are a major source of IL-10 that disrupts IFN-gamma activity in L. major-susceptible BALB/c mice. However, the increase in CD4(+)Foxp3(+) T cells within the IL-4Ralpha(-/-) dermis implies a possible IL-10-independent role for Treg cells within the infection site, and may indicate a novel immune escape mechanism used by L. major parasites in the absence of IL-4/IL-13 signaling.

Urea-amide preferential interactions in water: quantitative comparison of model compound data with biopolymer results using water accessible surface areas.

Two fundamentally different thermodynamic approaches are in use to interpret or predict the effects of urea on biopolymer processes: one is a synthesis of transfer free energies obtained from measurements of the effects of urea on the solubilities of small, model compounds; the other is an analysis of preferential interactions of urea with a range of folded and unfolded biopolymer surfaces. Here, we compare the predictions of these two approaches for the contribution of urea-amide (peptide) interactions to destabilization of folded proteins by urea. For these comparisons, we develop independent thermodynamic analyses of osmometric and solubility data characterizing interactions of a model compound with urea (or any other solute) and apply them to all five model compounds (glycine, alanine, diglycine, glycylalanine, and triglycine) where both isopiestic distillation (ID) and solubility data in aqueous urea solutions are available. We use model-independent expressions to calculate mu ex 23, the derivative of the "excess" chemical potential of solute "2" (either a model compound or a biopolymer) with respect to the molality of solute "3" (urea). Analyses of ID data for these systems reveal significant dependences of mu ex 23 on both m2 and m3, which must be taken into account in making comparisons with values of mu ex 23 obtained from solubility studies or from analyses of urea-biopolymer preferential interactions. Values of mu ex 23 calculated from model compound ID data at low m2 and m3 are directly proportional to the amount of polar amide (N, O) surface area, and not to any other type of surface. The proportionality constant in this limit, mu ex 23 /(RT x ASA) = (1.0 +/- 0.1) x 10(-3) A(-2), is very similar to that previously obtained by analysis of urea-biopolymer preferential interactions ((1.4 +/- 0.3) x 10(-3) A(-2)). This level of agreement for amide surface in the low concentration limit, as well as the absence of any significant preferential interaction of urea with Gly and Ala, reinforces the conclusion that the primary preferential interaction of urea with protein surface is a favorable interaction (resulting in local accumulation of urea) at polar amide surface, located mostly on the peptide backbone. However, mu ex 23 for interactions of urea with these model amides is found from both ID and solubility data to be urea concentration-dependent, in contrast to the urea concentration independence of the analogous quantity for protein unfolding.

CD4(+)CD25(-)Foxp3(-) Th1 cells are the source of IL-10-mediated immune suppression in chronic cutaneous leishmaniasis.

Nonhealing forms of leishmaniasis in humans are commonly associated with elevated levels of the deactivating cytokine IL-10, and in the mouse, normally chronic infections can be cleared in the absence of IL-10. Using a Leishmania major strain that produces nonhealing dermal lesions in a T helper type 1 (Th1) cell-polarized setting, we have analyzed the cellular sources of IL-10 and their relative contribution to immune suppression. IL-10 was produced by innate cells, as well as CD4(+)CD25(+)Foxp3(+) and CD4(+)CD25(-)Foxp3(-) T cells in the chronic lesion. Nonetheless, only IL-10 production by antigen-specific CD4(+)CD25(-)Foxp3(-) T cells, the majority of which also produced IFN-gamma, was necessary for suppression of acquired immunity in Rag(-/-) reconstituted mice. Surprisingly, Rag(-/-) mice reconstituted with naive CD4(+) T cells depleted of natural T regulatory cells developed more severe infections, associated with elevated levels of IL-10 and, especially, Th2 cytokines in the site. The data demonstrate that IL-10-producing Th1 cells, activated early in a strong inflammatory setting as a mechanism of feedback control, are the principal mediators of T cell-derived IL-10-dependent immune suppression in a chronic intracellular infection.

Nonhealing infection despite Th1 polarization produced by a strain of Leishmania major in C57BL/6 mice.

Experimental Leishmania major infection in mice has been of immense interest because it was among the first models to demonstrate the importance of the Th1/Th2 balance to infection outcome in vivo. However, the Th2 polarization that promotes the development of nonhealing cutaneous lesions in BALB/c mice has failed to adequately explain the mechanisms underlying nonhealing forms of leishmaniasis in humans. We have studied a L. major strain from a patient with nonhealing lesions that also produces nonhealing lesions with ulcerations and high parasite burden in conventionally resistant C57BL/6 mice. Surprisingly, these mice develop a strong, polarized, and sustained Th1 response, as evidenced by high levels of IFN-gamma produced by Leishmania-specific cells in the draining lymph node and in the ear lesion, and an absence of IL-4 or IL-13. The parasites fail to be effectively cleared despite high level induction of inducible NO synthase in the lesion, and despite their sensitivity to killing by IFN-gamma-activated macrophages in vitro. Infection of IL-10(-/-) mice, blockade of the IL-10R, or depletion of CD25(+) cells during the chronic phase promotes parasite killing, indicating that IL-10 and regulatory T cells play a role in rendering the Th1 responses ineffective at controlling infection in the skin. Mice with nonhealing primary lesions are nonetheless resistant to reinfection in the other ear. We suggest that nonhealing infections in animal models that are explained not by aberrant Th2 development, but by overactivation of homeostatic pathways designed to control inflammation, provide better models to understand nonhealing or reactivation forms of leishmaniasis in humans.

Gibbs-Duhem-based relationships among derivatives expressing the concentration dependences of selected chemical potentials for a multicomponent system.

For a two-component system, a derivative that specifies the concentration-dependence of one chemical potential can be calculated from the corresponding derivative of the other chemical potential by applying the Gibbs-Duhem Equation. To extend the practical utility of this binary thermodynamic linkage to systems having any number of components, we present a derivation based on a previously unrecognized recursive relationship. Thus, for each independently variable component, kappa, any derivative of its chemical potential, mukappa, with respect to one of the mole ratios {mkappa identical with nkappa/nomega} is related to as a characteristic series of progressively higher order derivatives of muomega for a single "probe" component, omega, with respect to certain of the {mkappa}. For aqueous solutions in which omega is solvent water and one or more of the solutes (kappa) is dilute, under typical conditions each sum of terms expressing a derivative of mukappa consists of at most a few numerically significant contributions, which can be quantified, or at least estimated, by analyzing osmometric data to determine how the single chemical potential muomega depends on the {mkappa} without neglecting any significant contributions from the other components. Expressions derived here also will provide explicit criteria for testing various approximations built into alternative analytic strategies for quantifying derivatives that specify the {mkappa} dependences of mukappa for selected components. Certain quotients of these derivatives are of particular interest in so far as they gauge important thermodynamic effects due to "preferential interactions".

The exclusion of glycine betaine from anionic biopolymer surface: why glycine betaine is an effective osmoprotectant but also a compatible solute.

Paradoxically, glycine betaine (N,N,N-trimethyl glycine; GB) in vivo is both an effective osmoprotectant (efficient at increasing cytoplasmic osmolality and growth rate) and a compatible solute (without deleterious effects on biopolymer function, including stability and activity). For GB to be an effective osmoprotectant but not greatly affect biopolymer stability, we predict that it must interact very differently with folded protein surface than with that exposed in unfolding. To test this hypothesis, we quantify the preferential interaction of GB with the relatively uncharged surface exposed in unfolding the marginally stable lacI helix-turn-helix (HTH) DNA binding domain using circular dichroism and with the more highly charged surfaces of folded hen egg white lysozyme (HEWL) and bovine serum albumin (BSA) using all-gravimetric vapor pressure osmometry (VPO) and compare these results with results of VPO studies (Hong et al. (2004), Biochemistry, 43, 14744-14758) of the interaction of GB with polyanionic duplex DNA. For these four biopolymer surfaces, we observe that the extent of exclusion of GB per unit of biopolymer surface area increases strongly with increasing fraction of anionic oxygen (protein carboxylate or DNA phosphate) surface. In addition, GB is somewhat more excluded from the surface exposed in unfolding the lacI HTH and from the folded surface of HEWL than expected from their small fraction of anionic surface, consistent with moderate exclusion of GB from polar amide surface, as predicted by the osmophobic model of protein stability (Bolen and Baskakov (2001) J. Mol. Biol. 310, 955-963). Strong exclusion of GB from anionic surface explains how it can be both an effective osmoprotectant and a compatible solute; analysis of this exclusion yields a lower bound on the hydration of anionic protein carboxylate surface of two layers of water (>or=0.22 H(2)O A(-)(2)).

Preferential interactions of glycine betaine and of urea with DNA: implications for DNA hydration and for effects of these solutes on DNA stability.

Interactions of the solutes glycine betaine (GB) and urea with mononucleosomal calf thymus DNA in aqueous salt solutions are characterized by vapor pressure osmometry (VPO). Analysis of osmolality as a function of solute and DNA concentration yields the effect of the solute on the chemical potential, mu(2), of the DNA. Although both GB and urea generally are nucleic acid denaturants and therefore must interact favorably with the nucleic acid surface exposed upon melting, VPO demonstrates that neither interacts favorably with duplex DNA. Addition of GB greatly increases mu(2) of DNA, indicating that the average local concentration of GB in the vicinity of the double helix is much less than its bulk concentration. By contrast, addition of urea has almost no effect on mu(2) of duplex DNA, indicating that the average local concentration of urea in the vicinity of duplex DNA is almost the same as in bulk solution. Qualitatively, we conclude that the nonuniform distribution of GB occurs primarily because duplex DNA and GB prefer to interact with water rather than with each other. Comparison with thermodynamic data for the interaction of GB with various protein surfaces (Felitsky et al., Biochemistry, 43, 14732-14743) shows that GB is excluded primarily from anionic DNA surface and that the hydration of anionic DNA phosphate oxygen surface (>or approximately 17 H(2)O per nucleotide or >or approximately 0.22 H(2)O A(-)(2)) involves at least two layers of water. From analysis of literature data for effects of urea and of GB on DNA melting, we propose that urea is an effective nonspecific nucleic acid denaturant because of its favorable interactions with the polar amide-like surface of G, C, and especially T or U bases exposed in denaturation, whereas GB is a specific GC denaturant because of its favorable interaction with G and/or C surface in the single-stranded state.

T cell biasing by activated dendritic cells.

We have previously reported that the ligation of FcgammaRs on activated macrophages affected their production of cytokines and their ability to influence T cell activation. Dendritic cells (DC) are important APCs that also express FcgammaR. In the present work, we sought to determine whether DC responded to immune complexes in a manner similar to macrophages. We confirmed that activated murine DC produced IL-12, and, as a result, induced naive T cells to produce primarily IFN-gamma upon stimulation. However, DC activated in the presence of immune complexes shut off their production of IL-12p70 and induced a Th2-like cytokine response. Thus, DC respond to immune complexes by altering their cytokine production, which, in turn, influences T cell responses. A DC transfer experiment was performed to determine the extent that APC exposure to immune complexes could influence adaptive immune responses. Vaccination of mice with Ag, along with DC that were activated in the presence of immune complexes, resulted in higher levels of Ag-specific IgG1 Ab, relative to mice that were vaccinated with activated DC and Ag alone. The mechanism by which DC altered their cytokine production in response to immune complexes was different from macrophages. Macrophages down-regulated the transcription of both the p40 and p35 subunits of IL-12, whereas DC decreased only p35 expression. We conclude that APCs expressing FcgammaR on their surface can respond to immune complexes by shutting off IL-12 biosynthesis, to prevent the Th1-type T cell biasing that normally accompanies innate immune activation.

Preferential interactions in aqueous solutions of urea and KCl.

A quantitative characterization of the thermodynamic effects due to interactions of salt ions and urea in aqueous solution is needed for rigorous analyses of the effects of changing urea concentration on biopolymer processes in solutions that also contain salt. Therefore, we investigate preferential interactions in aqueous solutions containing KCl and urea by using vapor pressure osmometry (VPO) to measure osmolality as a function of the molality of urea (component 3) over the range 0.09

Generalized derivation of an exact relationship linking different coefficients that characterize thermodynamic effects of preferential interactions.

In solutions consisting of solvent water (component '1') and two solute components ('2' and '3'), various thermodynamic effects of differences between solute-solute and solute-solvent interactions are quantitatively characterized by state functions commonly called 'preferential interaction coefficients': gamma(mu(1),mu(3)) triple bond (delta(m3)/delta(m2))(T,mu(1),mu(3)) and gamma(mu(k)) triple bond (delta(m3)/delta(m2))(T,P,mu(k)), where k = 1,2 or 3. These different derivatives are not all directly accessible to experimental determination, nor are they entirely equivalent for analyses and interpretations of thermodynamic and molecular effects of preferential interactions. Consequently, various practical and theoretical considerations arise when, for a given system, different kinds of preferential interaction coefficients have significantly different numerical values. Previously we derived the exact relationship linking all three coefficients of the type gamma(mu(k), and hence identified the physical origins of the differences between gamma(mu(1)) and gamma(mu(3)) that have been experimentally determined for each of various common biochemical solutes interacting with a protein [J. Phys. Chem. B, 106 (2002) 418-433]. Continuing our investigation of exact thermodynamic linkages among different types of preferential interaction coefficients, we present here a generalized derivation of the relationship linking gamma(mu(1),mu(3)), gamma(mu(3)) and gamma(mu(1)), with no restrictions on m(2), m(3) or any physical characteristic of either solute component (such as partial molar volume). Hence, we show that (gamma(mu(1),mu(3)) - gamma(mu(3))) is related directly to (gamma(mu(3)) - gamma(mu(1))), for which the physical determinants have been considered in detail previously, and to a factor dependent on the ratio of the partial molar volumes V3/V1. Our generalized expression also provides a basis for calculating gamma(mu(1),mu(3)), even in situations where preferential interactions could not be investigated by equilibrium dialysis. To demonstrate this applicability, we analyze isopiestic distillation data for aqueous solutions containing urea and NaCl, two small solute components that cannot be selectively dialyzed.

A novel phenotype for an activated macrophage: the type 2 activated macrophage.

Activated macrophages were used as antigen presenting cells (APCs) to determine the extent to which these APCs could influence an adaptive immune response. We show that activated macrophages induced a strong polarized Th1-like T cell response that was predominated by IFN-gamma. However, when antigen was targeted to Fcgamma receptors on these macrophages, their phenotype changed, and they now induced a T cell response that was predominated by IL-4. The initial biasing by activated macrophages toward a Th1-like response was a result of activation of the innate immune response, as macrophages from MyD88(-/-) mice failed to produce Th1-inducing cytokines. The reversal of the Th1 biasing was a result of FcgammaR ligation, as macrophages lacking the FcR common gamma chain failed to reverse this biasing. To show that this biasing could occur in vivo, mice were injected with activated macrophages or activated macrophages whose FcgammaR had been ligated with an irrelevant immune complex. Mice injected with FcgammaR-ligated macrophages made more antibody than those receiving conventionally activated macrophages, and the antibody was predominantly of the IgG1 isotype. These studies demonstrate that FcgammaR ligation on activated macrophages can change the phenotype of these APCs to cells that preferentially drive a Th2-like response. We have termed these cells type 2 activated macrophages.

Cutting edge: biasing immune responses by directing antigen to macrophage Fc gamma receptors.

An immune response can deviate toward either a Th1- or Th2-like response. In this work we examine the contribution that activated macrophages and IgG Abs make toward this deviation. The use of activated macrophages as APCs resulted in a strong polarized T cell response that was predominated by IFN-gamma. However, when Ag was targeted to FcgammaRs on these macrophages, the T cell response was reversed and biased toward a Th2-like response. This Th2-like phenotype was stable and was retained when the T cells were subsequently restimulated under nonbiasing conditions. The T cell biasing and its reversal via FcgammaR was also observed in vivo. Mice vaccinated with IgG-opsonized OVA made high levels of IgG Ab of the IgG1 isotype. These studies demonstrate that the ligation of FcgammaR on activated macrophages can reverse the Th1 biasing that occurs as a result of innate immune responses to microbial products.

Differential SLP-76 expression and TCR-mediated signaling in effector and memory CD4 T cells.

We present in this study novel findings on TCR-mediated signaling in naive, effector, and memory CD4 T cells that identify critical biochemical markers to distinguish these subsets. We demonstrate that relative to naive CD4 T cells, memory CD4 T cells exhibit a profound decrease in expression of the linker/adapter molecule SLP-76, while effector T cells express normal to elevated levels of SLP-76. The reduced level of SLP-76 is memory CD4 T cells is coincident with reduced phosphorylation overall, yet the residual SLP-76 couples to a subset of TCR-associated linker molecules, leading to downstream mitogen-activated protein (MAP) kinase activation. By contrast, effector CD4 T cells strongly phosphorylate SLP-76, linker for activation of T cells, and additional Grb2-coupled proteins, exhibit increased associations of SLP-76 to phosphorylated linkers, and hyperphosphorylate downstream Erk1/2 MAP kinases. Our results suggest distinct coupling of signaling intermediates to the TCR in naive, effector, and memory CD4 T cells. Whereas effector CD4 T cells amplify existing TCR signaling events accounting for rapid effector responses, memory T cells engage fewer signaling intermediates to efficiently link TCR triggering directly to downstream MAP kinase activation.

Modulating macrophage function with IgG immune complexes.

Macrophages respond to bacterial products by releasing a large array of inflammatory mediators. We demonstrate that, in the presence of IgG immune complexes, macrophages produce high levels of IL-10 and virtually no IL-12, when they are exposed to bacterial products. The production of IL-10 by these cells can dampen innate inflammatory responses to microbial products, such as LPS. This alteration in macrophage cytokine production can also influence an adaptive immune response, preferentially inducing Th2-type immunity. Thus, immune complexes change the physiology of activated macrophages, converting them to anti-inflammatory cells that induce Th2-like immune responses. We have termed these cells type II activated macrophages.