Abstract Using a mouse mutagenesis screen, we have identified CD83 as being critical for the development of CD4+ T cells and for their function postactivation. CD11c+ dendritic cells develop and function normally in mice with a mutated CD83 gene but CD4+ T cell development is substantially reduced. Additionally, we now show that those CD4+ cells that develop in a CD83 mutant animal fail to respond normally following allogeneic stimulation. This is at least in part due to an altered cytokine expression pattern characterized by an increased production of IL-4 and IL-10 and diminished IL-2 production. Thus, in addition to its role in selection of CD4+ T cells, absence of CD83 results in the generation of cells with an altered activation and cytokine profile.
Bispecific antibodies (bsAbs) present an attractive opportunity to combine the additive and potentially synergistic effects exhibited by combinations of monoclonal antibodies (mAbs). Current challenges for engineering bsAbs include retention of the binding affinity of the parent mAb or antibody fragment, the ability to bind both targets simultaneously, and matching valency with biology. Other factors to consider include structural stability and expression of the recombinant molecule, both of which may have significant impact on its development as a therapeutic. Here, we incorporate selection of stable, potent single-chain variable fragments (scFvs) early in the engineering process to assemble bsAbs for therapeutic applications targeting the cytokines IL-17A/A and IL-23. Stable scFvs directed against human cytokines IL-23p19 and IL-17A/A were isolated from a human Fab phage display library via batch conversion of panning output from Fabs to scFvs. This strategy integrated a step for shuffling V regions during the conversion and permitted the rescue of scFv molecules in both the V(H)V(L) and the V(L)V(H) orientations. Stable scFvs were identified and assembled into several bispecific formats as fusions to the Fc domain of human IgG1. The engineered bsAbs are potent neutralizers of the biological activity of both cytokines (IC(50) < 1 nM), demonstrate the ability to bind both target ligands simultaneously and display stability and productivity advantageous for successful manufacture of a therapeutic molecule. Pharmacokinetic analysis of the bsAbs in mice revealed serum half-lives similar to human mAbs. Assembly of bispecific molecules using stable antibody fragments offers an alternative to reformatting mAbs and minimizes subsequent structure-related and manufacturing concerns.
A combined university/industry team has developed a prototype system for handling protein crystals aboard the space station. This system uses a miniature direct drive robot, CCD television cameras, and a client-server computing system using Internet protocols to support the capture of protein crystals from aqueous growth solutions. The system was demonstrated between Huntsville AL. and Seattle WA. An operator in Huntsville controlled the mini robot by invoking predefined relative and absolute macro files. The operators observed results using video images sent through the Internet link using Cu-SeeMe video conferencing software. In 3 of 4 trials, the operators successfully captured 0.5 mm simulated protein crystals into a glass capillary. The system is a promising start for the development of a space-station based remote protein crystal analysis facility.
The proinflammatory cytokines IL-17A and IL-17F have a high degree of sequence similarity and share many biological properties. Both have been implicated as factors contributing to the progression of inflammatory and autoimmune diseases. Moreover, reagents that neutralize IL-17A significantly ameliorate disease severity in several mouse models of human disease. IL-17A mediates its effects through interaction with its cognate receptor, the IL-17 receptor (IL-17RA). We report here that the IL-17RA-related molecule, IL-17RC is the receptor for IL-17F. Notably, both IL-17A and IL-17F bind to IL-17RC with high affinity, leading us to suggest that a soluble form of this molecule may serve as an effective therapeutic antagonist of IL-17A and IL-17F. We generated a soluble form of IL-17RC and demonstrate that it effectively blocks binding of both IL-17A and IL-17F, and that it inhibits signaling in response to these cytokines. Collectively, our work indicates that IL-17RC functions as a receptor for both IL-17A and IL-17F and that a soluble version of this protein should be an effective antagonist of IL-17A and IL-17F mediated inflammatory diseases.
Abstract The scarcity of effective biomarkers and therapeutic strategies for predicting disease onset and progression in Alzheimer’s disease (AD) is a major challenge to improve much-needed therapeutic outcomes. Conventional drug discovery approaches have been unsuccessful in providing efficient interventions due to their ‘one-size-fits-all’ nature. As an alternative, personalised drug development holds promise to pre-select responders and identify suitable drug efficacy indicators. In this study, we established a preclinical drug testing strategy by assessing the efficacy of anti-inflammatory drugs in 2D and 3D in vitro models of monocyte-derived microglia-like cells (MDMi) derived from AD and mild cognitive impairment (MCI) patients, and matched healthy individuals. We observed that the cytokine inflammatory profiles of MDMi in response to drugs clustered separately between cohorts, with the 3D model showing a more defined separation between healthy and patient donors than 2D. By ranking donor and cytokine responses to drugs, we identified that drug efficacy was limited in AD patients and involved cohort-specific responsive cytokines. Our findings suggest that MDMi models have the potential to predict disease progression, stratify responders and identify biomarkers for estimating the efficacy of microglia-targeted drugs. Together, our pipeline could serve as a valuable tool to enhance the clinical translational value of preclinical drug screens and ultimately improve drug outcomes for AD.
Epidermal kemtinocytes grow clonally when provided with a 3T3 feeder tayer and medium supplemented witb 20% foetal bovine serum, hydrocortisone and cholera toxin. In this culture system the proliferative response of freshly isolated human and mouse keratinocytes to a short exposure (24 h) of the tumour promoter phorbol, 12-myristate, 13-acetate (PMA) was dependent on the donor age but was independent of the species or the biopsy site. Human keratinocytes from early (16–18 week) foetal donors displayed a strong proliferative response to PMA as determined by a 5- to 7-fold increase in colony number and an increase in the average colony size. In contrast, adult and juvenile keratinocytes of all ages from both mice and humans displayed an inhibition of colony forming efficiency and a reduction in colony size. When continuously passaged in culture human foetal keratinoctes gradually changed the pattern of their response to PMA so that they were inhibited from growing rather than being stimulated and after 21 days (three passages) their response was quantitatively similar to juvenile keratioocytes. Co-culture of juvenile keratinocytes with irradiated foetal keratinocytes did not alter their response to PMA so that the observed proliferative response of foetal keratinocytes to PMA is not readilly explained by the autsecretion of mitogens or other regulatory molecules by these cells. Late foetal (17 days gestation), noeonatal and post-neonatal (5–10 days old) mouse keratinocytes were also inhibited from growing by PMA but the magnitude of the effect was directly related to the age of the mouse and was in all cases less than that observed with adult mice. Tbe relationship of these results to the mechanisn of action of phorbol esters in epidermis is discussed.
A human cervical keratinocyte cell line, W12, has been initiated from a low-grade cervical lesion histologically diagnosed as CIN I. This cell line has, to date, undergone over 300 generations in vitro with an average doubling time of 30 hr: an aneuploid karyotype has developed with progressive in vitro growth. W12 contains HPV16 DNA present at approximately 100 copies and the state of the viral DNA over a number of passages has been examined. The HPV16 DNA is stably maintained at high copy number over several passages and restriction enzyme analysis together with electrophoresis of uncleaved viral DNA indicate that it is present predominantly as the episomal molecule. W12 cells exhibit a typical keratinocyte morphology and, when transplanted into the flank of the nude mouse, form an epithelial lesion with the histological features of CIN I/II.
