Impact of Targeting Moiety Type and Protein Corona Formation on the Uptake of Fn14-Targeted Nanoparticles by Cancer Cells.

The TWEAK receptor, Fn14, is a promising candidate for active targeting of cancer nanotherapeutics to many solid tumor types, including metastatic breast and primary brain cancers. Targeting of therapeutic nanoparticles (NPs) has been accomplished using a range of targeting moieties including monoclonal antibodies and related fragments, peptides, and small molecules. Here, we investigated a full-length Fn14-specific monoclonal antibody, ITEM4, or an ITEM4-Fab fragment as a targeting moiety to guide the development of a clinical formulation. We formulated NPs with varying densities of the targeting moieties while maintaining the decreased nonspecific adhesivity with receptor targeting (DART) characteristics. To model the conditions that NPs experience following intravenous infusion, we investigated the impact of serum exposure in relation to the targeting moiety type and surface density. To further evaluate performance at the cancer cell level, we performed experiments to assess differences in cellular uptake and trafficking in several cancer cell lines using confocal microscopy, imaging flow cytometry, and total internal reflection fluorescence microscopy. We observed that Fn14-targeted NPs exhibit enhanced cellular uptake in Fn14-high compared to Fn14-low cancer cells and that in both cell lines uptake levels were greater than observed with control, nontargeted NPs. We found that serum exposure increased Fn14-targeted NP specificity while simultaneously reducing the total NP uptake. Importantly, serum exposure caused a larger reduction in cancer cell uptake over time when the targeting moiety was an antibody fragment (Fab region of the monoclonal antibody) compared with the full-length monoclonal antibody targeting moiety. Lastly, we uncovered that full monoclonal antibody-targeted NPs enter cancer cells via clathrin-mediated endocytosis and traffic through the endolysosomal pathway. Taken together, these results support a pathway for developing a clinical formulation using a full-length Fn14 monoclonal antibody as the targeting moiety for a DART cancer nanotherapeutic agent.

Complicated Diverticulitis: Age Distribution, Management and Burden on Health Care.

INTRODUCTION: Acute diverticulitis represents a common surgical condition and one of the leading gastrointestinal causes of hospital admissions in Western societies. The aim of the study is to examine the distribution, management and cost to healthcare of complicated diverticulitis and compare those to uncomplicated diverticulitis. METHODS: The case-control study was performed for patients with acute diverticulitis in Lyell McEwin Hospital in Adelaide, South Australia. Data were collected for patients presented from January 2015 to December 2017. Consecutive patients with acute diverticulitis confirmed by computed tomography were included in the study. Patients recruited for the study were divided into two groups. Patients presenting with Hinchey Ia diverticulitis were classified as 'uncomplicated diverticulitis'. Patients who presented with Hinchey Ib, II, III or IV diverticulitis were classified as 'complicated diverticulitis'. The Hinchey classification was based on the radiological reports of CT scans. RESULTS: From 2015-2017, 116 cases were screened for the study, 10 of which were excluded due to not having CT diagnosis. A total of 106 consecutive cases of acute diverticulitis were recruited for the study. Forty-four cases had complicated diverticulitis. Sixty-two cases with uncomplicated diverticulitis were allocated as a control group. The distribution of cases spanned through all age groups. There were nine cases (20.9%) in the 30-39 age group in the complicated diverticulitis compared to eight cases (12.9%) in the uncomplicated group with odds ratio 1.7 (0.61-4.92). The mean length of stay of the complicated diverticulitis group was 7.74 days compared to 3.93 days of the uncomplicated group with a p value of 0.000235. Nine (20%) cases of the 44 complicated diverticulitis cases were managed operatively, while 35 (80%) of the complicated diverticulitis group and all of the uncomplicated (control) group were managed conservatively. Localized perforations were 24 cases (54.5%) of the complicated diverticulitis group and collections were 18 cases (40.8%). Those cases collectively represented the majority of the complicated group. CONCLUSION: Complicated diverticulitis increases the length of stay significantly in acute diverticulitis cases that are requiring hospital admission despite conservative management in 80% of the cases. Younger age groups represent a significant percentage of both complicated and uncomplicated diverticulitis. In the study population, the percentage of the younger age group was higher in complicated diverticulitis compared to uncomplicated diverticulitis, although this increased risk did not reach statistical significance. This will need to be further investigated in future studies.

Evaluation of Clinical Assessment in Predicting Complicated Acute Diverticulitis.

Background Acute diverticulitis is a common surgical condition and one of the leading gastrointestinal conditions that require hospital admission. The presence of complications increases the hospital stay and risk of requiring surgical intervention. This study aimed to investigate the clinical features that can be identified during clinical assessment and evaluate their predictive value and sensitivity in differentiating between complicated and uncomplicated diverticulitis. Methodology This retrospective case-control study was performed on patients with acute diverticulitis at Lyell McEwin Hospital, Adelaide, South Australia. Data were collected for patients presenting from January 2015 to December 2017. Patients with acute diverticulitis confirmed by computed tomography (CT) were included in the study. Multiple clinical assessment aspects were reported and compared between complicated diverticulitis and uncomplicated diverticulitis groups. Results Data from a total of 116 cases were collected, 10 of which were excluded due to lack of CT diagnosis. Forty-four cases had complicated diverticulitis (case group), and 62 cases had uncomplicated diverticulitis (control group). Twenty-three cases (52.2%) had the first episode of diverticulitis in the complicated group compared to 24 cases (38.7%) in the uncomplicated group, with an odds ratio of 1.73 (0.79-3.789). Eight cases (18.2%) had previously complicated diverticulitis in the complicated group compared to 11 cases (17.7%) in the uncomplicated group, with an odds ratio of 1.03 (0.37-2.82). Six cases (13.6%) had a fever (T > 38) in the complicated group compared to two cases (3.2%) in the uncomplicated group, with an odds ratio of 4.74 (0.9-24.7), a sensitivity of only 13.64%, and a specificity of 96.77%. Twelve cases (27.3%) had tachycardia, two cases (4.5%) had hypotension, and five cases (11.4%) had peritonism in the complicated group compared to two cases (3.2%), one case (1.6%), and one case (1.6%) in the uncomplicated group, with odds ratios of 11.25 (2.37-53.4), 2.9 (0.255-33), and 7.82 (0.88-69.5), respectively; sensitivity was 27.27%, 4.55%, and 11.36% for tachycardia, hypotension, and peritonism, whereas specificity was 96.77%, 98.39%, and 98.39%, respectively. Conclusions The study found no significant correlation between having complicated diverticulitis and previous episodes of complicated diverticulitis, immunosuppression, pain severity, or change in bowel habits. Perrectal bleeding was found to reduce the risk of having complicated diverticulitis. Our results did not demonstrate a statistically significant relationship between the first episode of diverticulitis and having complicated diverticulitis. Physical signs, when abnormal, are highly specific in predicting complicated diverticulitis. Tachycardia was found to have the highest positive predictive value and odds ratio compared to the other observed physical signs.

Functional communication between IP3R and STIM2 at subthreshold stimuli is a critical checkpoint for initiation of SOCE.

Stromal interaction molecules, STIM1 and STIM2, sense decreases in the endoplasmic reticulum (ER) [Ca2+] ([Ca2+]ER) and cluster in ER-plasma membrane (ER-PM) junctions where they recruit and activate Orai1. While STIM1 responds when [Ca2+]ER is relatively low, STIM2 displays constitutive clustering in the junctions and is suggested to regulate basal Ca2+ entry. The cellular cues that determine STIM2 clustering under basal conditions is not known. By using gene editing to fluorescently tag endogenous STIM2, we report that endogenous STIM2 is constitutively localized in mobile and immobile clusters. The latter associate with ER-PM junctions and recruit Orai1 under basal conditions. Agonist stimulation increases immobile STIM2 clusters, which coordinate recruitment of Orai1 and STIM1 to the junctions. Extended synaptotagmin (E-Syt)2/3 are required for forming the ER-PM junctions, but are not sufficient for STIM2 clustering. Importantly, inositol 1,4,5-triphosphate receptor (IP3R) function and local [Ca2+]ER are the main drivers of immobile STIM2 clusters. Enhancing, or decreasing, IP3R function at ambient [IP3] causes corresponding increase, or attenuation, of immobile STIM2 clusters. We show that immobile STIM2 clusters denote decreases in local [Ca2+]ER mediated by IP3R that is sensed by the STIM2 N terminus. Finally, under basal conditions, ambient PIP2-PLC activity of the cell determines IP3R function, immobilization of STIM2, and basal Ca2+ entry while agonist stimulation augments these processes. Together, our findings reveal that immobilization of STIM2 clusters within ER-PM junctions, a first response to ER-Ca2+ store depletion, is facilitated by the juxtaposition of IP3R and marks a checkpoint for initiation of Ca2+ entry.

STIM2 targets Orai1/STIM1 to the AKAP79 signaling complex and confers coupling of Ca2+ entry with NFAT1 activation.

The Orai1 channel is regulated by stromal interaction molecules STIM1 and STIM2 within endoplasmic reticulum (ER)-plasma membrane (PM) contact sites. Ca2+ signals generated by Orai1 activate Ca2+-dependent gene expression. When compared with STIM1, STIM2 is a weak activator of Orai1, but it has been suggested to have a unique role in nuclear factor of activated T cells 1 (NFAT1) activation triggered by Orai1-mediated Ca2+ entry. In this study, we examined the contribution of STIM2 in NFAT1 activation. We report that STIM2 recruitment of Orai1/STIM1 to ER-PM junctions in response to depletion of ER-Ca2+ promotes assembly of the channel with AKAP79 to form a signaling complex that couples Orai1 channel function to the activation of NFAT1. Knockdown of STIM2 expression had relatively little effect on Orai1/STIM1 clustering or local and global [Ca2+]i increases but significantly attenuated NFAT1 activation and assembly of Orai1 with AKAP79. STIM1ΔK, which lacks the PIP2-binding polybasic domain, was recruited to ER-PM junctions following ER-Ca2+ depletion by binding to Orai1 and caused local and global [Ca2+]i increases comparable to those induced by STIM1 activation of Orai1. However, in contrast to STIM1, STIM1ΔK induced less NFAT1 activation and attenuated the association of Orai1 with STIM2 and AKAP79. Orai1-AKAP79 interaction and NFAT1 activation were recovered by coexpressing STIM2 with STIM1ΔK. Replacing the PIP2-binding domain of STIM1 with that of STIM2 eliminated the requirement of STIM2 for NFAT1 activation. Together, these data demonstrate an important role for STIM2 in coupling Orai1-mediated Ca2+ influx to NFAT1 activation.

Heterogeneous Origin of Gonadotropin Releasing Hormone-1 Neurons in Mouse Embryos Detected by Islet-1/2 Expression.

In vertebrates, Gonadotropin releasing hormone-1 (GnRH) neuroendocrine cells originate in the olfactory placode and migrate into the forebrain where they regulate reproduction. However, the embryonic lineage of their progenitors remains controversial. Most GnRH neurons are derived from placodal ectodermal progenitor cells, but data from lineage tracing in zebrafish (Whitlock et al., 2003) and mouse (Forni and Wray, 2012) indicate that some GnRH progenitor cells have a neural crest (NC) origin. In contrast, a recent study in zebrafish (Aguillon et al., 2018), using Islet-1/2 expression, identified this LIM-homeodomain protein in all developing GnRH neuroendocrine cells, and the authors concluded a homogenous origin from progenitors within the preplacodal ectoderm. Evidence in different animal models and systems suggests that expression of Islet-1 plays a pivotal role in cell fate specification and differentiation. Thus, expression of Islet-1/2 in all GnRH cells in the nasal placode may not be lineage dependent but rather initiated locally in the placode as part of the program for GnRH cell specification and/or differentiation. This study addresses this issue and shows two populations of olfactory derived GnRH neurons in embryonic mouse: Islet-1/2(+) and Islet-1/2(-). Notably, triple-label immunofluorescence using the NC lineage tracer Wnt1, showed that GnRH neurons derived from Wnt1 progenitors are Islet-1/2(-). These results are consistent with two separate origins of GnRH neuroendocrine cells and suggest that either (1) NC-derived GnRH cells differentiate earlier than PE-derived GnRH cells or (2) different programs are used for cell specification in NC- vs. PE-derived GnRH cells.

GPR37 Signaling Modulates Migration of Olfactory Ensheathing Cells and Gonadotropin Releasing Hormone Cells in Mice.

Gonadotropin releasing hormone (GnRH) neurons, part of the hypothalamic-pituitary-gonadal axis, regulate reproduction. Prenatally, GnRH neurons migrate into the brain from the nasal placode along terminal nerve fibers, intermixed with olfactory sensory axons and olfactory ensheathing cells (OECs). An expression analysis from embryonic GnRH neurons identified the G protein-coupled receptor 37 (GPR37 or PAEL-r). GPR37 has been linked to (1) juvenile Parkinson's disease in humans, (2) oligodendrocyte differentiation, and (3) Wnt/ß-catenin signaling during neurogenesis. In this study, the role of GPR37 was investigated in the developing GnRH/olfactory system. PCR and immunocytochemistry confirmed expression of GPR37 in migrating GnRH neurons as well as in OECs. Inhibition of GPR37 signaling in nasal explants attenuated GnRH neuronal migration and OEC movement. Examination of GPR37 deficient mice revealed a decrease in the olfactory bulb nerve layer and attenuated/delayed maturation and migration of GnRH neurons into the brain. These data demonstrate a developmental role for GPR37 signaling in neural migration. SIGNIFICANCE STATEMENT: Reproduction is controlled by gonadotrophin releasing hormone (GnRH) neurons located in the central nervous system. Embryonically, GnRH neurons originate in the nasal/olfactory placode and migrate into the brain on axonal tracks from cells in the vomeronasal organ, intermixed with olfactory sensory axons and olfactory ensheathing cells (OECs). An expression analysis from embryonic GnRH neurons identified the G protein-coupled receptor 37. Here we show that inhibition of GPR37 signaling in nasal explants and mutant mice attenuated GnRH neuronal migration. Signaling via GPR37 also perturbed OEC movement, resulting in a decrease in the olfactory bulb nerve layer in vivo. Together, these results identify a new role for GPR37 signaling during development - modulating cell migration.

Reelin Can Modulate Migration of Olfactory Ensheathing Cells and Gonadotropin Releasing Hormone Neurons via the Canonical Pathway.

One key signaling pathway known to influence neuronal migration involves the extracellular matrix protein Reelin. Typically, signaling of Reelin occurs via apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor (VLDLR), and the cytoplasmic adapter protein disabled 1 (Dab1). However, non-canonical Reelin signaling has been reported, though no receptors have yet been identified. Cariboni et al. (2005) indicated Dab1-independent Reelin signaling impacts gonadotropin releasing hormone-1 (GnRH) neuronal migration. GnRH cells are essential for reproduction. Prenatal migration of GnRH neurons from the nasal placode to the forebrain, juxtaposed to olfactory axons and olfactory ensheathing cells (OECs), has been well documented, and it is clear that alterations in migration of these cells can cause delayed or absent puberty. This study was initiated to delineate the non-canonical Reelin signaling pathways used by GnRH neurons. Chronic treatment of nasal explants with CR-50, an antibody known to interfere with Reelin homopolymerization and Dab1 phosphorylation, decreased the distance GnRH neurons and OECs migrated. Normal migration of these two cell types was observed when Reelin was co-applied with CR-50. Immunocytochemistry was performed to determine if OECs might transduce Reelin signals via the canonical pathway, and subsequently indirectly altering GnRH neuronal migration. We show that in mouse: (1) both OECs and GnRH cells express ApoER2, VLDLR and Dab1, and (2) GnRH neurons and OECs show a normal distribution in the brain of two mutant reeler lines. These results indicate that the canonical Reelin pathway is present in GnRH neurons and OECs, but that Reelin is not essential for development of these two systems in vivo.