Control of therapeutic IgG antibodies galactosylation during cultivation process and its impact on IgG1/FcγR interaction and ADCC activity.

Therapeutic monoclonal antibodies (mAbs) are within the fastest growing group of pharmaceuticals on the global market. IgG1 subclass is the most potent effector in Fc-related functions. The N-linked glycosylation of mAbs Fc-domain significantly influences its therapeutic activity and the presence of this modification is largely dependent on producer cell and parameters of manufacturing process. Here we examined and characterized cell culture conditions that determine during cultivation selective changes in galactose content of a model therapeutic mAb IgG1, trastuzumab biosimilar. We demonstrated that such in cultivation process shift of galactosylation does not affect binding of the mAb to its antigen yet modifies interaction of the mAb with Fcγ receptors and therefore enhances antibody dependent cellular cytotoxicity (ADCC).

Recombinant C1 Esterase Inhibitor Reduces Cytokine Storm in an Ex Vivo Whole Blood Model.

C1 esterase inhibitor (C1INH) is an abundant component of blood plasma (the average concentration is 250 mg/L); it is known to be involved in several biological processes, for instance, in the regulation of the coagulation system, adhesion of leukocytes on endothelial cells, and in the regulation of complement and kallikrein cascades. Lately, the role of C1INH in immunomodulation has gained considerable attention. We used an ex vivo whole blood model to examine the influence of C1INH and its mutated variants on the inflammatory cytokines interleukin (IL)-6, tumor necrosis factor-α (TNFα), and IL-1ß. The present study demonstrated for the first time that recombinant C1INH or its Seprin domain can downregulate bacterial endotoxin induced IL-6 release. We also observed that unstructured N-terminal domain of C1INH downregulates the release of IL-1ß and TNFα, but not IL-6. Our results suggest that C1INH may have therapeutic potential for treatment of inflammatory conditions.

Application of RNAi technology and fluorescent protein markers to study membrane traffic in C. elegans.

RNA interference (RNAi) is a powerful tool to study the intracellular membrane transport and membrane organelle behavior in the nematode Caenorhabditis elegans. This model organism has gained popularity in the trafficking field because of its relative simplicity, yet being multicellular. C. elegans is fully sequenced and has an annotated genome, it is easy to maintain, and a growing number of transgenic strains bearing markers for different membrane compartments are available. C. elegans is particularly well suited for protein downregulation by RNAi because of the simple but efficient methods of dsRNA delivery. The phenomenon of systemic RNAi in the worm further facilitates this approach. In this chapter we describe methods and applications of RNAi in the field of membrane traffic. We summarize the fluorescent markers used as a readout for the effects of gene knockdown in different cells and tissues and give details for data acquisition and analysis.

Targeting the Wnt pathways for therapies.

The Wnt/ß-catenin signaling pathway is crucial in animal development from sponges to humans. Its activity in the adulthood is less general, with exceptions having huge medical importance. Namely, improper activation of this pathway is carcinogenic in many tissues, most notably in the colon, liver and the breast. On the other hand, the Wnt/ß-catenin signaling must be re-activated in cases of tissue damage, and insufficient activation results in regeneration failure and degeneration. These both medically important implications are unified by the emerging importance of this signaling pathway in the control of proliferation of various types of stem cells, crucial for tissue regeneration and, in case of cancer stem cells - cancer progression and relapse. This article aims at briefly reviewing the current state of knowledge in the field of Wnt signaling, followed by a detailed discussion of current medical developments targeting distinct branches of the Wnt pathway for anti-cancer and pro-regeneration therapies.

Identification of the switch in early-to-late endosome transition.

Sequential transport from early to late endosomes requires the coordinated activities of the small GTPases Rab5 and Rab7. The transition between early and late endosomes could be mediated either through transport carriers or by Rab conversion, a process in which the loss of Rab5 from an endosome occurs concomitantly to the acquisition of Rab7. We demonstrate that Rab conversion is the mechanism by which proteins pass from early to late endosomes in Caenorhabditis elegans coelomocytes. Moreover, we identified SAND-1/Mon1 as the critical switch for Rab conversion in metazoa. SAND-1 serves a dual role in this process. First, it interrupts the positive feedback loop of RAB-5 activation by displacing RABX-5 from endosomal membranes; second, it times the recruitment of RAB-7, probably through interaction with the HOPS complex to the same membranes. SAND-1/Mon1 thus acts as a switch by controlling the localization of RAB-5 and RAB-7 GEFs.

Application of RNAi technology and fluorescent protein markers to study membrane traffic in Caenorhabditis elegans.

Ribonucleic acid interference (RNAi) is a powerful tool for study of the intracellular membrane transport and membrane organelle behavior in the nematode Caenorhabditis elegans. This model organism has gained popularity in the trafficking field because of its relative simplicity, yet multicellularity. Caenorhabditis elegans is fully sequenced and has an annotated genome, it is easy to maintain, and a growing number of transgenic strains bearing markers for different membrane compartments are available. Caenorhabditis elegans is particularly well suited for protein downregulation by RNAi because of the simple but efficient methods of double-stranded RNA (dsRNA) delivery. The phenomenon of systemic RNAi in the worm further facilitates this approach. In this chapter, we describe methods and applications of RNAi in the field of membrane traffic. We summarize the fluorescent markers used as a readout for the effects of gene knockdown in different cells and tissues and give details for data acquisition and analysis.

A proGDNF-related peptide BEP increases synaptic excitation in rat hippocampus.

The glial cell-derived neurotrophic factor (GDNF) precursor contains several putative sites for prohormone convertase-mediated excision of short peptides. Here, we show that one of the predicted peptides, named BEP (brain excitatory peptide), induces a substantial increase in the synaptic excitability in rat CA1 pyramidal neurons. The excitation is sensitive to N-ethylmaleimide, suggesting involvement of a G-protein-coupled receptor.

Cortical granule exocytosis in C. elegans is regulated by cell cycle components including separase.

In many organisms, cortical granules undergo exocytosis following fertilization, releasing cargo proteins that modify the extracellular covering of the zygote. We identified cortical granules in Caenorhabditis elegans and have found that degranulation occurs in a wave that initiates in the vicinity of the meiotic spindle during anaphase I. Previous studies identified genes that confer an embryonic osmotic sensitivity phenotype, thought to result from abnormal eggshell formation. Many of these genes are components of the cell cycle machinery. When we suppressed expression of several of these genes by RNAi, we observed that cortical granule trafficking was disrupted and the eggshell did not form properly. We conclude that osmotic sensitivity phenotypes occur because of defects in trafficking of cortical granules and the subsequent formation of an impermeable eggshell. We identified separase as a key cell cycle component that is required for degranulation. Separase localized to cortically located filamentous structures in prometaphase I upon oocyte maturation. After fertilization, separase disappeared from these structures and appeared on cortical granules by anaphase I. RNAi of sep-1 inhibited degranulation in addition to causing extensive chromosomal segregation failures. Although the temperature-sensitive sep-1(e2406) allele exhibited similar inhibition of degranulation, it had minimal effects on chromosome segregation. These observations lead us to speculate that SEP-1 has two separable yet coordinated functions: to regulate cortical granule exocytosis and to mediate chromosome separation.

Caenorhabditis elegans SAND-1 is essential for RAB-7 function in endosomal traffic.

The small rab-GTPase RAB-7 acts in endosome and endosome to lysosome traffic. We identified SAND-1 as a protein required for RAB-7 function based on similarities between SAND-1 and RAB-7 RNAi phenotypes. Although the initial uptake of yolk protein in oocytes, or of soluble secreted (ss) GFP in coelomocytes, appeared normal, further transport along the endocytic traffic route was delayed in the absence of SAND-1 function, and yolk proteins failed to reach yolk granules efficiently. Moreover, in coelomocytes, ssGFP and BSA-Texas-Red were endocytosed but not transported to lysosomes. We show that SAND-1 is essential for RAB-7 function at the transition from early to late endosomes, but not for RAB-7 function at lysosomes.

Involvement of the actin cytoskeleton and homotypic membrane fusion in ER dynamics in Caenorhabditis elegans.

The endoplasmic reticulum (ER) is the major intracellular membrane system. The ER is essential for protein and lipid biosynthesis, transport of proteins along the secretory pathway, and calcium storage. Here, we describe our investigations into the dynamics and regulation of the ER in the early Caenorhabditis elegans embryo. Using a GFP fusion to the ER-resident signal peptidase SP12, we observed the morphological transitions of the ER through fertilization and the early cell-cycles in living embryos. These transitions were tightly coordinated with the division cycle: upon onset of mitosis, the ER formed structured sheets that redispersed at the initiation of cleavage. Although microtubules were not required for the transition of the ER between these different states, the actin cytoskeleton facilitated the dispersal of the ER at the end of mitosis. The ER had an asymmetric distribution in the early embryo, which was dependent on the establishment of polarity by the PAR proteins. The small GTPase ARF-1 played an essential role in the ER dynamics, although this function appeared to be unrelated to the role of ARF-1 in vesicular traffic. In addition, the ER-resident heat shock protein BiP and a homologue of the AAA ATPase Cdc48/p97 were found to be crucial for the ER transitions. Both proteins have been implicated in homotypic ER membrane fusion. We provide evidence that homotypic membrane fusion is required to form the sheet structure in the early embryo.

GDNF promotes tubulogenesis of GFRalpha1-expressing MDCK cells by Src-mediated phosphorylation of Met receptor tyrosine kinase.

Glial cell line-derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are multifunctional signaling molecules in embryogenesis. HGF binds to and activates Met receptor tyrosine kinase. The signaling receptor complex for GDNF typically includes both GDNF family receptor alpha1 (GFRalpha1) and Ret receptor tyrosine kinase. GDNF can also signal independently of Ret via GFRalpha1, although the mechanism has remained unclear. We now show that GDNF partially restores ureteric branching morphogenesis in ret-deficient mice with severe renal hypodysplasia. The mechanism of Ret-independent effect of GDNF was therefore studied by the MDCK cell model. In MDCK cells expressing GFRalpha1 but no Ret, GDNF stimulates branching but not chemotactic migration, whereas both branching and chemotaxis are promoted by GDNF in the cells coexpressing Ret and GFRalpha1, mimicking HGF/Met responses in wild-type MDCK cells. Indeed, GDNF induces Met phosphorylation in several ret-deficient/GFRalpha1-positive and GFRalpha1/Ret-coexpressing cell lines. However, GDNF does not immunoprecipite Met, making a direct interaction between GDNF and Met highly improbable. Met activation is mediated by Src family kinases. The GDNF-induced branching of MDCK cells requires Src activation, whereas the HGF-induced branching does not. Our data show a mechanism for the GDNF-induced branching morphogenesis in non-Ret signaling.