Gregory Kanter

Expression of Active Murine Granulocyte‐Macrophage Colony‐Stimulating Factor in an Escherichia coli Cell‐Free System

Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) is an important cytokine in the mammalian immune system. It has been expressed in Escherichia coli with the same biological activity as the native protein. Here, we report the synthesis of a murine recombinant GM‐CSF in an E. coli cell‐free protein synthesis system with a high yield. Since there are two disulfide bonds in the native structure of GM‐CSF, an oxidizing redox potential of the reaction mixture was required. By pretreating the cell extract with iodoacetamide (IAM), the reducing activity of the cell extract was inactivated, and upon further application of an oxidized glutathione buffer, most of the synthesized GM‐CSF was found in its oxidized form. However, the GM‐CSF thus formed showed low activity because of poor folding. With the addition of DsbC, the periplasmic disulfide isomerase from E. coli, a high yield of active GM‐CSF was produced in the cell‐free reaction. Finally, successful folding of the cell‐free synthesized GM‐CSF‐his6 was confirmed by its cell‐proliferation activity after purification with a Ni2+ chelating column.

Placenta accreta in a patient with a history of uterine artery embolization for postpartum hemorrhage

Uterine artery embolization (UAE) is used to treat various conditions from uterine leiomyoma to uncontrollable bleeding. We describe a case of placenta accreta after a prior delivery, which required UAE to control a postpartum hemorrhage. This case highlights the importance of both antenatal evaluation of placentation and heightened precaution for delivery in subsequent pregnancies for women who have undergone this procedure.

Urogynecology digest : Presented by Gregory Kanter.

This population-based prospective cohort study followed 3,435 participants aged 65 or older sampled randomly from the Seattle Group Heath (GH) healthcare system between the years 1994 and 2012. Using GH pharmacy records, a calculated total standardized daily dose (TSDD) of anticholinergic exposure took into account the dose of a medication in relation to its minimum effective dose, and was used to determine whether cumulative anticholinergic dose over a 10-year period is associated with the development of dementia. In primarily white participants with amedian age of 74.4 years at entry and amean follow-up of 7.3 years, 797 participants (23.2 %) developed dementia, 79.9 % of whom had probable Alzheimer’s dementia (AD). The authors found a dose–response relationship of increased dementia risk with increased anticholinergic use. This association became significant only at the highest cumulative dose, with an adjusted hazard ratio (95 % CI) of 1.63 (1.24– 2.14), adjusting for sex, age at entry, and APOE-4 genotype. To safeguard against protopathic bias (that medications were being given for a preexisting condition such as depression, which might precede dementia), recent medication use that may have indicated prodromal symptoms was eliminated from analysis, and analysis of anticholinergic subtype showed no difference between antidepressants and other anticholinergic classes. This well-designed study is timely given the high rate of anticholinergic use for a variety of indications in our aging population, and the prospective design allowed for standardized screening for and definitions of dementia. This, combined with long-term accurate accounts of anticholinergic exposure, provide robust data for looking at the effect of anticholinergic exposure on dementia risk. Because past anticholinergic use had a similar dementia risk to continuing use, it would appear that effects persist despite medication discontinuation. The authors cite increased AD in Parkinson’s patients with greater anticholinergic exposure and elevatedβ-amyloid concentration in mice with reduced cholinergic transmission to give biological plausibility to this relationship. Generalizability is limited by a relatively homogeneous patient population and the potential misclassification of use due to over the counter medications and patients possibly not taking prescriptions that were filed. Of note, the effects of anticholinergics on dementia, though statistically significant, were modest, and high-potency anticholinergics such as doxepin, chlorpheniramine, and oxybutynin were necessary to obtain the highest level of exposure. Nonetheless, these results add to existing studies to suggest an increased risk of dementia with 3 years of exposure to a high-potency anticholinergic, which warrants caution and discussion with patients about the potential risks of long-term use.