Therapeutic proteins like human interferon a2 generally possess short serum half-lives due to their small size, hence rapid renal clearance, and susceptibility to serum proteases. Chemical derivatization, such as addition of polyethylene glycol (PEG) groups overcomes both problems, but at the expense of greatly decreased bioactivity. We describe a new method that yields biologically potent interferon a2b (IFNa2) in high yields and with increased serum half-life when expressed as arabinogalactan-protein (AGP) chimeras in cultured tobacco cells. Thus IFNa2-AGPs targeted for secretion typically gave 350-1400-fold greater secreted yields than the non-glycosylated IFNa2 control. The purified AGP domain itself was not immunogenic when injected into mice and only mildly so when injected as a fusion glycoprotein. Importantly, the AGPIFNa2 chimeras showed up to a 13-fold increased in vivo serum half-life while the biological activity remained similar to native IFNa2. The use of arabinogalactan glycomodules may provide a general approach to the enhanced production of therapeutic proteins by plants.

Therapeutic proteins with molecular weights lower than 40 kDa often have short serum half-lives due to their susceptibility to serum proteases and rapid renal clearance. Chemical derivatization, such as PEGylation, or expression as serum albumin fusions increases molecular mass and overcome these problems but at the expense of decreased bioactivity. Here we applied a new method that yields biologically potent recombinant human growth hormone (rhGH) with increased serum half-life when expressed as an arabinogalactan-protein (AGP) in tobacco BY-2 cells. Thus, rhGH was expressed with 10 repeats of the AGP glycomodule Ser-Hyp (SO) at the C-terminus (rhGH-(SO)(10)). We also expressed rhGH as an AGP-enhanced green fluorescent protein (EGFP) fusion, designated rhGH-(SO)(10)-EGFP, to assess the cellular distribution of the glycoprotein, which was mainly extracellular. Recombinant hGH-(SO)(10) bound the hGH receptor with an affinity similar to that of a rhGH standard, stimulated the same intracellular signaling pathway as hGH, but possessed an in vivo serum half-life more than sixfold that of the hGH control. Furthermore, rhGH-(SO)(10) gave a 500 fold greater secreted yield than the non-glycosylated control rhGH that was also targeted for secretion. Detailed analysis of the rhGH-(SO)(10) glycans indicated a conserved structure with relatively little microheterogeneity and an average size of 25 monosaccharide residues. These results were consistent with earlier work expressing interferon alpha 2b as an AGP chimera and further demonstrate the feasibility of this approach to the production of long-acting, biologically potent therapeutic proteins by plant cells.

It has been claimed that osteopathic manipulative treatment (OMT) is able to enhance the immune response of individuals. In particular, it has been reported that OMT has the capability to increase antibody titers, enhance the efficacy of vaccination, and upregulate the numbers of circulating leukocytes. Recently, it has been shown in human patients suffering chronic low back pain, that OMT is able to modify the levels of cytokines such as IL-6 and TNF-α in blood upon repeated treatment. Further, experimental animal models show that lymphatic pump techniques can induce a transient increase of cytokines in the lymphatic circulation. Taking into account all these data, we decided to investigate in healthy individuals the capacity of OMT to induce a rapid modification of the levels of cytokines and leukocytes in circulation. Human volunteers were subjected to a mixture of lymphatic and thoracic OMT, and shortly after the levels of several cytokines were evaluated by protein array technology and ELISA multiplex analysis, while the profile and activation status of circulating leukocytes was extensively evaluated by multicolor flow cytometry. In addition, the levels of nitric oxide and C-reactive protein (CRP) in plasma were determined. In this study, our results show that OMT was not able to induce a rapid modification in the levels of plasma nitrites or CRP or in the proportion or activation status of central memory, effector memory or naïve CD4 and CD8 T cells. A significant decrease in the proportion of a subpopulation of blood dendritic cells was detected in OMT patients. Significant differences were also detected in the levels of immune molecules such as IL-8, MCP-1, MIP-1α and most notably, G-CSF. Thus, OMT is able to induce a rapid change in the immunological profile of particular circulating cytokines and leukocytes.

The role of complement receptor type 3 (CR3) in nonopsonic recognition of group B streptococci (GBS) by macrophages was investigated. Monoclonal anti-CR3 (anti-Mac-1) inhibited phagocytosis of GBS strains by as much as 50% in serum-free cultures of both mouse peritoneal macrophages and the macrophage cell line PU5-1.8. GBS uptake was unaffected by the presence of anti-C3 or salicylhydroxamate, an inhibitor of the covalent binding reaction of C3. Soluble antibodies to LFA-1 or to the common beta-chain (CD18) of the LFA-1/CR3/p150,95 family of cell adhesion molecules did not inhibit GBS uptake. Down-modulation of surface Mac-1 on macrophages following adherence to anti-Mac-1- or anti-CD18-coated surfaces also inhibited uptake of GBS. Further evidence for GBS interaction with CR3 was demonstrated by reduction of EC3bi rosette formation in macrophages adherent to GBS-coated plates. These studies suggest that GBS can interact with macrophage CR3, promoting phagocytosis in a C3-independent fashion. In the absence of specific immunity in neonates, this recognition mechanism may be a significant virulence determinant for GBS which poorly activate the alternate complement pathway.

C3H/HeJ mice were used to study the origin and nature of endotoxin-induced glucocorticoid antagonizing factor (GAF). In conventional mice GAF is believed to be responsible for a variety of effects that occur as a result of an injection of endotoxin, including the inhibition of hormonal induction of hepatic phosphoenolpyruvate carboxykinase and of glyconeogenesis. Responses in such animals are seen whether the endotoxin is extracted with phenol-water or with trichloroacetic acid. C3H/HeJ mice do not respond (or produce GAF?) after an intravenous injection of phenol-water lipopolysaccharide, but they react normally (produce GAF?) when given a trichloroacetic acid preparation. They also behave the same as conventional animals when injected with serum from poisoned normal mice, especially when the reticuloendothelial system of the donors has been activated by prior injections of Zymosan or heat-killed tubercle bacilli. The C3H/HeJ mice have been used, therefore, as assay animals to establish that peak levels of GAF appear in donor serum about 2 h after an injection of lipopolysaccharide, and it is produced intraperitoneally in C3H/HeJ mice given a mixture of endotoxin and peritoneal exudate cells derived from responder mice. GAF elutes from Sephadex G-200 along with markers of known molecular weight in the region of 100,000 to 200,000. It is inactivated by trypsin and by heating at 75 degrees C for 1 h.