Neurofilament light as a predictive biomarker of unresolved chemotherapy-induced peripheral neuropathy in subjects receiving paclitaxel and carboplatin.

Management of chemotherapy-induced peripheral neuropathy (CIPN) remains a significant challenge in the treatment of cancer. Risk mitigation for CIPN involves preemptive reduction of cumulative dose or reduction of dose intensity upon emergence of symptoms, despite the risk of reduced tumor efficacy. A predictive biomarker for dose-limiting CIPN could improve treatment outcomes by allowing providers to make informed decisions that balance both safety and efficacy. To identify a predictive biomarker of CIPN, markers of neurodegeneration neurofilament-light (NfL), glial fibrillary acidic protein (GFAP), tau and ubiquitin c-terminal hydrolase L1 (UCHL1) were assessed in serum of up to 88 subjects drawn 21 days following the first of 6 treatments with chemotherapeutics paclitaxel and carboplatin. Serum NfL and GFAP were increased with chemotherapy. Further, NfL change predicted subsequent onset of grade 2-3 CIPN during the remainder of the trial (mean treatment duration = 200 days) and trended toward stronger prediction of CIPN that remained unresolved at the end of the study. These results confirm previous reports that serum NfL is increased in CIPN and provide the first evidence that NfL can be used to identify subjects susceptible to dose-limiting paclitaxel and carboplatin induced CIPN prior to onset of symptoms.

Outer segment phagocytosis by cultured retinal pigment epithelial cells requires Gas6.

The function and viability of vertebrate photoreceptors requires the daily phagocytosis of photoreceptor outer segments (OS) by the adjacent retinal pigment epithelium (RPE). We demonstrate here a critical role in this process for Gas6 and by implication one of its receptor protein tyrosine kinases (RTKs), Mertk (Mer). Gas6 specifically and selectively stimulates the phagocytosis of OS by normal cultured rat RPE cells. The magnitude of the response is dose-dependent and shows an absolute requirement for calcium. By contrast the Royal College of Surgeons (RCS) rat RPE cells, in which a mutation in the gene Mertk results in the expression of a truncated, non-functional receptor, does not respond to Gas6. These data strongly suggest that activation of Mertk by its ligand, Gas6, is the specific signaling pathway responsible for initiating the ingestion of shed OS. Moreover, photoreceptor degeneration in the RCS rat retina, which lacks Mertk, and in humans with a mutation in Mertk, strongly suggests that the Gas6/Mertk signaling pathway is essential for photoreceptor viability. We believe that this is the first demonstration of a specific function for Gas6 in the eye.

Further studies on the identification of the phagocytosis receptor of rat retinal pigment epithelial cells.

We have previously produced a polyclonal antiserum (R1S5) against a plasma membrane-enriched fraction of rat retinal pigment epithelial (RPE) cells which inhibits the phagocytosis of photoreceptor outer segments (OS) by these cells. This antiserum has now been used to purify a subset of RPE membrane glycoproteins. Using a combination of lectin affinity chromatography, and chromatography on an affinity column made with R1S5-IgG, we have enriched an RPE membrane extract about 100-fold. This enriched extract contains only 12 components, all of which are glycoproteins, and retains the ability to adsorb out the inhibitory activity of antiserum R1S5. This shows that one or more of these glycoproteins recognizes an inhibitory IgG in R1S5 and suggests that one or more of these glycoproteins may participate in the phagocytosis of OS by RPE cells, possibly as the phagocytosis receptor. We have performed N-terminal microsequencing of seven of these glycoproteins: four of the seven, with Mrs of 34, 36, 51 and 55 kDa, show no sequence homology to any known proteins.

Carbachol does not correct the defect in the phagocytosis of outer segments by Royal College of Surgeons rat retinal pigment epithelial cells.

PURPOSE: To investigate the effect of carbachol on the phagocytosis of photoreceptor outer segments (OS) in cultures of normal Long-Evans and dystrophic Royal College of Surgeons (RCS) rat retinal pigment epithelial (RPE) cells. METHODS: Retinal pigment epithelial cells from normal and RCS rats were grown in tissue culture. On reaching confluence, they were presented with OS suspended in Krebs-Henseleit buffer in the presence or absence of carbachol and LiCl. The number of bound and ingested OS was quantitated using double immunofluorescence staining. RESULTS: LiCl inhibited the ingestion of OS by more than 90% but had no effect on the binding of OS by Long-Evans RPE cells. The addition of carbachol further reduced OS ingestion. Carbachol alone decreased OS ingestion by normal RPE cells by 30% but had no effect on OS binding. The effect of LiCl and carbachol on RCS RPE cells was similar to their effect on normal RPE cells. CONCLUSIONS: Carbachol does not increase OS phagocytosis in normal or RCS rat RPE cells. The phagocytic defect in RCS rat RPE cannot be reversed or overcome by stimulation of the IP3 pathway by carbachol. LiCl strongly inhibits the ingestion of OS by normal and by RCS RPE cells, and this effect is enhanced by carbachol.

The effect of inhibitors of glycoprotein synthesis and processing on the phagocytosis of rod outer segments by cultured retinal pigment epithelial cells.

Retinal pigment epithelial cells selectively phagocytize rod outer segments by a process that may be mediated by specific cell surface receptors. Since many receptors are glycoproteins, we have studied the effect of tunicamycin, an inhibitor of N-linked oligosaccharide synthesis, and of castanospermine and swainsonine, which are inhibitors of oligosaccharide processing, on the ability of cultured retinal pigment epithelial cells to phagocytize rod outer segment. Tunicamycin inhibits the glycosylation of newly synthesized glycoproteins by 85-90%; concomitantly, the phagocytosis of rod outer segments is inhibited by 70-80%. The effect of tunicamycin is to initially reduce rod outer segments binding, and therefore the subsequent ingestion of rod outer segments. SDS-PAGE analysis and autoradiography of [35S]methionine labelled extracts of tunicamycin-treated cells, demonstrates the disappearance of a number of glycoprotein bands, and the appearance of a number of protein bands of lower Mr. Kinetic analysis of the disappearance and reappearance of specific glycoproteins suggests that the lower Mr bands are the non-glycosylated forms of the higher Mr bands. By contrast, castanospermine and swainsonine have no effect on the ability of retinal pigment epithelial cells to phagocytize rod outer segments, or on the SDS-PAGE pattern of treated cells, although they were shown to inhibit oligosaccharide processing as expected. These results support the hypothesis that rod outer segment phagocytosis by retinal pigment epithelial cells is mediated by specific glycoprotein receptors. N-Glycosylation of these receptors is required for their function, or for their insertion into the plasma membrane, whereas processing of the N-linked oligosaccharide chains of these receptors is not crucial for rod outer segment phagocytosis by retinal pigment epithelial cells.