Preventing Postoperative Atypical Mycobacterial Infection.

PURPOSE: To report a recent outbreak of atypical mycobacterial infection following upper eyelid surgery and the results of a prevention protocol that was successfully instituted to dramatically reduce the infection rate. METHODS: This is a multicenter retrospective nonrandomized comparative interventional case series. A chart review of 7 patients who developed atypical mycobacterium infection after undergoing blepharoplasty was conducted. Preventative intervention included exchanging ice compresses for reusable gel packs and substituting tap water with bottled or distilled water for facial cleaning during postoperative care. The main outcome measure was disease incidence. RESULTS: Of the 368 patients who underwent upper eyelid blepharoplasty and/or blepharoptosis repair from December 2014 to May 2015, 7 people developed cutaneous atypical mycobacterium infection with an incidence of 1.9%. Eighty-six percent of patients received clarithromycin as part of their treatment with average treatment length of 2.8 months. Debridement was performed in 71% of the patients. Biopsy was performed in all patients, and all had histopathology showing granulomatous inflammation. A prevention protocol was developed to reduce potential inoculation in the immediate postoperative period, which successfully reduced the infection rate from 1.9% to 0.06% (p = 0.019). CONCLUSIONS: Atypical mycobacterium infection, although rare, should be considered as a possible diagnosis in a blepharoplasty patient with delayed development of nodular lesions. Long-term clarithromycin therapy and debridement have shown good outcomes for these patients; however, the best treatment for any infection is prevention. This study provides the first evidence based approach within the ophthalmic literature for reducing the mycobacterium infection rate in blepharoplasty patients.

The missense of smell: functional variability in the human odorant receptor repertoire.

Humans have ~400 intact odorant receptors, but each individual has a unique set of genetic variations that lead to variation in olfactory perception. We used a heterologous assay to determine how often genetic polymorphisms in odorant receptors alter receptor function. We identified agonists for 18 odorant receptors and found that 63% of the odorant receptors we examined had polymorphisms that altered in vitro function. On average, two individuals have functional differences at over 30% of their odorant receptor alleles. To show that these in vitro results are relevant to olfactory perception, we verified that variations in OR10G4 genotype explain over 15% of the observed variation in perceived intensity and over 10% of the observed variation in perceived valence for the high-affinity in vitro agonist guaiacol but do not explain phenotype variation for the lower-affinity agonists vanillin and ethyl vanillin.

A protocol for heterologous expression and functional assay for mouse pheromone receptors.

Innate social behaviors like intermale aggression, fear, and mating rituals are important for survival and propagation of a species. In mice, these behaviors have been implicated to be mediated by peptide pheromones that are sensed by a class of G protein-coupled receptors, vomeronasal receptor type 2 (V2Rs), expressed in the pheromone-detecting vomeronasal organ (VNO) (Chamero et al., Nature 450:899-902, 2007; Haga et al., Nature 466:118-122, 2010; Kimoto et al., Curr Biol 17:1879-1884, 2007; Leinders-Zufall et al., Nat Neurosci 12:1551-1558, 2009; Papes et al., Cell 141:692-703, 2010). Matching V2Rs with their cognate ligands is required to understand what receptors the biologically relevant pheromones are acting on. However, this goal has been greatly limited by the unavailability of appropriate heterologous tools commonly used to carry out receptor deorphanization, due to the fact that this family of receptors fails to traffic to the surface of heterologous cells. We have demonstrated that calreticulin, a housekeeping chaperone commonly expressed in most eukaryotic cells, is sparsely expressed in the vomeronasal sensory neurons (VSNs). Stable knock down of calreticulin in a HEK293T derived cell line (R24 cells) allows us to functionally express V2Rs on the surface of heterologous cells. In this chapter we describe protocols for maintenance and expansion of the R24 cell line and functional assays for V2Rs using these cells.

Assaying surface expression of chemosensory receptors in heterologous cells.

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors (GPCRs) that are postulated to bind volatile odorant molecules and converting the extracellular signal into an intracellular signal by coupling with G protein Gαolf. Binding of the odorants to the receptors is thought to follow a combinatorial rule, that is, one odorant may bind several receptors and one receptor may bind several odorants to varying degrees. Biochemical, signaling and ligand binding studies have been conveniently carried out for most GPCRs using heterologous cells. However use of heterologous cells for study of odorant receptors, was precluded for a long time since on transfection they failed to export to the surface. Saito et al have demonstrated single membrane pass Receptor Transporting Protein (RTP) family chaperones show enhanced expression in the olfactory sensory neurons and act as chaperones to traffic odorant receptors to the surface in heterologous cells, when co transfected together. To carry out biochemical assays for receptors using heterologous cells, one must first determine if the receptor shows robust surface expression in the cell line. This can be assayed by overexpressing the receptors with the chaperone RTP1S followed by live cell staining to fluorescently label the extracellular domain or a tag in the extracellular domain exclusively. Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.