Modular complement assemblies for mitigating inflammatory conditions.

Complement protein C3dg, a key linkage between innate and adaptive immunity, is capable of stimulating both humoral and cell-mediated immune responses, leading to considerable interest in its use as a molecular adjuvant. However, the potential of C3dg as an adjuvant is limited without ways of controllably assembling multiple copies of it into vaccine platforms. Here, we report a strategy to assemble C3dg into supramolecular nanofibers with excellent compositional control, using ß-tail fusion tags. These assemblies were investigated as therapeutic active immunotherapies, which may offer advantages over existing biologics, particularly toward chronic inflammatory diseases. Supramolecular assemblies based on the Q11 peptide system containing ß-tail-tagged C3dg, B cell epitopes from TNF, and the universal T cell epitope PADRE raised strong antibody responses against both TNF and C3dg, and prophylactic immunization with these materials significantly improved protection in a lethal TNF-mediated inflammation model. Additionally, in a murine model of psoriasis induced by imiquimod, the C3dg-adjuvanted nanofiber vaccine performed as well as anti-TNF monoclonal antibodies. Nanofibers containing only ß-tail-C3dg and lacking the TNF B cell epitope also showed improvements in both models, suggesting that supramolecular C3dg, by itself, played an important therapeutic role. We observed that immunization with ß-tail-C3dg caused the expansion of an autoreactive C3dg-specific T cell population, which may act to dampen the immune response, preventing excessive inflammation. These findings indicate that molecular assemblies displaying C3dg warrant further development as active immunotherapies.

Fusion of fibroblast growth factor 21 to a thermally responsive biopolymer forms an injectable depot with sustained anti-diabetic action.

Fibroblast growth factor 21 (FGF21) is under investigation as a type 2 diabetes protein drug, but its efficacy is impeded by rapid in vivo clearance and by costly production methods. To improve the protein's therapeutic utility, we recombinantly expressed FGF21 as a fusion with an elastin-like polypeptide (ELP), a peptide polymer that exhibits reversible thermal phase behavior. Below a critical temperature, ELPs exist as miscible unimers, while above, they associate into a coacervate. The thermal responsiveness of ELPs is retained upon fusion to proteins, which has notable consequences for the production and in vivo delivery of FGF21. First, the ELP acts as a solubility enhancer during E. coli expression, yielding active fusion protein from the soluble cell lysate fraction and eliminating the protein refolding steps that are required for purification of FGF21 from inclusion bodies. Second, the ELP's phase transition behavior is exploited for facile chromatography-free purification of the ELP-FGF21 fusion. Third, the composition and molecular weight of the ELP are designed such that the ELP-FGF21 fusion undergoes a phase transition triggered solely by body heat, resulting in an immiscible viscous phase upon subcutaneous (s.c.) injection and thereby creating an injectable depot. Indeed, a single s.c. injection of ELP-FGF21 affords up to five days of sustained glycemic control in ob/ob mice. The ELP fusion partner massively streamlines production and purification of FGF21, while providing a controlled release method for delivery that reduces the frequency of injection, thereby enhancing the pharmacological properties of FGF21 as a protein drug to treat metabolic disease.

Arginase Is Essential for Survival of Leishmania donovani Promastigotes but Not Intracellular Amastigotes.

Studies of Leishmania donovani have shown that both ornithine decarboxylase and spermidine synthase, two enzymes of the polyamine biosynthetic pathway, are critical for promastigote proliferation and required for maximum infection in mice. However, the importance of arginase (ARG), the first enzyme of the polyamine pathway in Leishmania, has not been analyzed in L. donovani To test ARG function in intact parasites, we generated Δarg null mutants in L. donovani and evaluated their ability to proliferate in vitro and trigger infections in mice. The Δarg knockout was incapable of growth in the absence of polyamine supplementation, but the auxotrophic phenotype could be bypassed by addition of either millimolar concentrations of ornithine or micromolar concentrations of putrescine or by complementation with either glycosomal or cytosolic versions of ARG. Spermidine supplementation of the medium did not circumvent the polyamine auxotrophy of the Δarg line. Although ARG was found to be essential for ornithine and polyamine synthesis, ornithine decarboxylase appeared to be the rate-limiting enzyme for polyamine production. Mouse infectivity studies revealed that the Δarg lesion reduced parasite burdens in livers by an order of magnitude but had little impact on the numbers of parasites recovered from spleens. Thus, ARG is essential for proliferation of promastigotes but not intracellular amastigotes. Coupled with previous studies, these data support a model in which L. donovani amastigotes readily salvage ornithine and have some access to host spermidine pools, while host putrescine appears to be unavailable for salvage by the parasite.

Controlled release of biologics for the treatment of type 2 diabetes.

Type 2 diabetes is a rapidly growing disease that poses a significant burden to the United States healthcare system. Despite the many available treatments for the disease, close to half of diagnosed type 2 diabetes cases are not properly managed, largely due to inadequate patient adherence to prescribed treatment regimens. Methods for improving delivery - and thereby easing administration - of type 2 drugs have the potential to greatly improve patient health. This review focuses on two peptide drugs - insulin and glucagon-like peptide 1 (GLP-1) - for treatment of type 2 diabetes. Peptide drugs offer the benefits of high potency and specificity but pose a significant delivery challenge due to their inherent instability and short half-life. The development of insulin and GLP-1 analogs highlights the broad spectrum of drug delivery strategies that have been used to solve these problems. Numerous structural modifications and formulations have been introduced to optimize absorption, residence time, stability, route of delivery and frequency of administration. Continual improvements in delivery methods for insulin and GLP-1 receptor agonists are paving the way towards better patient compliance and improved disease management, and thereby enhanced patient quality of life.

Genetic dissection of pyrimidine biosynthesis and salvage in Leishmania donovani.

Protozoan parasites of the Leishmania genus express the metabolic machinery to synthesize pyrimidine nucleotides via both de novo and salvage pathways. To evaluate the relative contributions of pyrimidine biosynthesis and salvage to pyrimidine homeostasis in both life cycle stages of Leishmania donovani, individual mutant lines deficient in either carbamoyl phosphate synthetase (CPS), the first enzyme in pyrimidine biosynthesis, uracil phosphoribosyltransferase (UPRT), a salvage enzyme, or both CPS and UPRT were constructed. The Δcps lesion conferred pyrimidine auxotrophy and a growth requirement for medium supplementation with one of a plethora of pyrimidine nucleosides or nucleobases, although only dihydroorotate or orotate could circumvent the pyrimidine auxotrophy of the Δcps/Δuprt double knockout. The Δuprt null mutant was prototrophic for pyrimidines but could not salvage uracil or any pyrimidine nucleoside. The capability of the Δcps parasites to infect mice was somewhat diminished but still robust, indicating active pyrimidine salvage by the amastigote form of the parasite, but the Δcps/Δuprt mutant was completely attenuated with no persistent parasites detected after a 4-week infection. Complementation of the Δcps/Δuprt clone with either CPS or UPRT restored infectivity. These data establish that an intact pyrimidine biosynthesis pathway is essential for the growth of the promastigote form of L. donovani in culture, that all uracil and pyrimidine nucleoside salvage in the parasite is mediated by UPRT, and that both the biosynthetic and salvage pathways contribute to a robust infection of the mammalian host by the amastigote. These findings impact potential therapeutic design and vaccine strategies for visceral leishmaniasis.

IMP dehydrogenase deficiency in Leishmania donovani causes a restrictive growth phenotype in promastigotes but is not essential for infection in mice.

Leishmania cannot synthesize purines de novo and therefore must scavenge purines from its host for survival and growth. Biochemical and genomic analyses have indicated that Leishmania species express three potential routes for the synthesis of guanylate nucleotides: (1) a two-step pathway that converts IMP to GMP; (2) a three-step pathway that starts with the deamination of guanine to xanthine, followed by phosphoribosylation to XMP and then conversion to GMP; or (3) direct guanine phosphoribosylation by HGPRT. To determine the role of the first of these pathways to guanylate nucleotide synthesis, an L. donovani line deficient in IMP dehydrogenase (IMPDH), the first step in the IMP to GMP pathway, was constructed by targeted gene replacement. The Δimpdh lesion triggered a highly restrictive growth phenotype in promastigotes in culture but did not impact parasitemias in mice. The dispensability of IMPDH in vivo is the first definitive demonstration that intracellular L. donovani amastigotes have access to a sufficient pool of guanine, xanthine, or guanylate precursors from the host.

Spermidine synthase is required for virulence of Leishmania donovani.

Genetic lesions in the polyamine biosynthetic pathway of Leishmania donovani, the causal agent of visceral leishmaniasis, are conditionally lethal mutations that render the insect vector form of the parasite auxotrophic for polyamines. Recently, we have demonstrated that a Δodc L. donovani null mutant lacking ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, was profoundly compromised in its ability to infect mice, indicating that ODC is essential for the infectious mammalian stage of the parasite and further validating the enzyme as a possible drug target. To assess whether other components of the polyamine biosynthetic pathway were also essential for parasite virulence, a cell line deficient in spermidine synthase (SPDSYN), the enzyme that converts putrescine to spermidine, was created by double-targeted gene replacement within a virulent L. donovani background. This Δspdsyn strain was auxotrophic for polyamines, required spermidine for growth in its insect vector form, and was adversely impacted in its ability to infect mice. These findings establish that SPDSYN, like ODC, is essential for maintaining a robust infection in mammals and indicate that pharmacologic inhibition of SPDSYN, and perhaps all components of the polyamine biosynthetic pathway, is a valid therapeutic strategy for the treatment of visceral and, potentially, other forms of leishmaniasis.

Linking NE1545 gene expression with cell volume changes in Nitrosomonas europaea cells exposed to aromatic hydrocarbons.

Nitrosomonas europaea, a model ammonia oxidizing bacterium, was exposed to a wide variety of aromatic hydrocarbons in 3 h batch assays. The expression of NE1545, a phenol sentinel gene involved in fatty acid metabolism, was monitored via quantitative real-time polymerase chain reaction (qRT-PCR) and a Coulter Counter technique was used to monitor changes in cell volume. Decreases in cell volume and NE1545 gene expression correlated strongly with exposure to aromatic hydrocarbons that possessed a single polar group substitution (e.g. phenol and aniline). Aromatic hydrocarbons that contain no polar group substitutions (e.g. toluene) or multiple polar group substitutions (e.g. p-hydroquinone) caused negligible changes in NE1545 expression and cell volume. The oxidation of aromatic hydrocarbons by N. europaea from configurations without a single polar group to one with two polar groups (e.g. p-cresol oxidized to 4-hydroxybenzyl alcohol) and from configurations with no polar groups to one with a single polar group (e.g. ethylbenzene oxidized to 4-ethylphenol) greatly influenced NE1545 gene expression and observed changes in cell volume. Nitrification inhibition in N. europaea by the aromatic hydrocarbons was found to be completely reversible; however, the decreases in cell volume were not reversible suggesting a physical change in cell membrane composition. Ammonia monooxygenase blocking studies showed that the chemical exposure that was responsible for the cell volume decrease and up-regulation in gene expression and not the observed inhibition. N. europaea is the first bacterium shown to experience significant changes in cell volume when exposed to µM concentrations of aromatic hydrocarbons, three orders of magnitude lower than previous studies with other bacteria.

Oral putrescine restores virulence of ornithine decarboxylase-deficient Leishmania donovani in mice.

Administration of putrescine as a 1% solution in the drinking water ameliorated the profound loss of virulence exhibited by ornithine decarboxylase (ODC) deficient Leishmania donovani in mice. Furthermore, supplying α-difluoromethylornithine, an ODC inhibitor, at 2% in the drinking water reduced but did not eliminate infection with wild type L. donovani in the mouse model. Taken collectively, these findings: (1) demonstrate that oral putrescine can access the phagolysosome of macrophages in which the parasite resides in mice; (2) establish that the loss of virulence due to the Δodc lesion is a consequence of the inability of the mutant parasite to synthesize sufficient polyamines de novo; (3) imply that the L. donovani amastigote cannot access host polyamines in sufficient amounts for survival and growth; (4) and validate ODC as a drug target, although oral administration of DFMO is an unlikely therapeutic paradigm for visceral leishmaniasis.