Immunodetection of NUT Protein: Implementation, Indications, and Results in a Tertiary Reference Center.

The immunodetection of NUT protein is a reliable tool to identify NUT carcinoma, a rare and still underdiagnosed tumor entity. The technique was implemented in 2017 in our department, a tertiary reference center with a large recruitment in all tumor types, including head and neck and thoracic tumors. We evaluated its use over a 6-year period (2017-2022) to (a) describe the indications for the technique, (b) determine the number of NUT carcinomas detected and confirmed by Fluorescence in situ hybridization, and (c) describe briefly the characteristics of these tumors. Over the study period, 382 NUT immunodetections were performed; the annual number of requests varied from 45 to 83. All 21 pathologists of the department made at least one request (range: 1 to 94; annual mean: 18.2). 54.7% of immunodetections were performed for internal cases, 37% for cases submitted for consultation, and 8.3% for cases submitted for confirmation of a suspected diagnosis. The main indications were poorly differentiated tumors of the head and neck region (39%) and the thorax (19.6%), and difficult-to-classify soft tissue tumors (11.8%). Twelve cases of NUT carcinoma were detected by immunohistochemistry and confirmed by Fluorescence in situ hybridization. Seven were from the head and neck region (4.7% of the tumors tested), 4 from lung or mediastinum (5.3%), 1 from an unknown primary at the time of diagnosis. In conclusion, the implementation of NUT immunodetection in the daily workflow of a pathology department improves the detection of NUT carcinoma. This becomes essential with the emergence of potential targeted therapies.

Link N as a therapeutic agent for discogenic pain.

Neurotrophins (NTs) are the major contributors of sensory axonal sprouting, neural survival, regulation of nociceptive sensory neurons, inflammatory hyperalgesia, and neuropathic pain. Intervertebral disc (IVD) cells constitutively express NTs. Their expression is upregulated by proinflammatory cytokines present in the IVD during degeneration, which can promote peripheral nerve ingrowth and hyperinnervation, leading to discogenic pain. Currently, there are no targeted therapies that decrease hyperinnervation in degenerative disc disease. Link N is a naturally occurring peptide with a high regenerative potential in the IVD. Therefore, the suitability of Link N as a therapeutic peptide for suppressing NTs, which are known modulators and mediators of pain, was investigated. The aim of the present study is to determine the effect of Link N on NTs expression, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and their cognate receptors TrkA and TrkB as they are directly correlated with symptomatic back pain. Furthermore, the neurotransmitter (substance P) was also evaluated in human annulus fibrosus (AF) cells stimulated with cytokines. Human AF cells isolated from normal IVDs were stimulated with interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in the presence or absence of Link N. NGF release in the media was evaluated by Western blotting. Total RNA was isolated and gene expression was measured using real-time PCR. Gene expression of NGF, BDNF, TrkA, and TrkB significantly decreased in human disc cells stimulated with either IL-1ß or TNF-α supplemented with Link N when compared to the cells stimulated only with IL-1ß or TNF-α. NGF protein expression was also suppressed in AF cells coincubated with Link N and IL-1ß when compared to the cells stimulated only with IL-1ß. Link N can suppress the stimulation of NGF, BDNF, and their receptors TrkA and TrkB in AF cells in an inflammatory milieu. Thus, coupled with previous observations, this suggests that administration of Link N has the potential to not only repair the discs in early stages of the disease but also suppress pain.

Naproxen induces type X collagen expression in human bone-marrow-derived mesenchymal stem cells through the upregulation of 5-lipoxygenase.

Several studies have shown that type X collagen (COL X), a marker of late-stage chondrocyte hypertrophy, is expressed in mesenchymal stem cells (MSCs) from osteoarthritis (OA) patients. We recently found that Naproxen, but not other nonsteroidal anti-inflammatory drugs (NSAIDs) (Ibuprofen, Celebrex, Diclofenac), can induce type X collagen gene (COL10A1) expression in bone-marrow-derived MSCs from healthy and OA donors. In this study we determined the effect of Naproxen on COL X protein expression and investigated the intracellular signaling pathways that mediate Naproxen-induced COL10A1 expression in normal and OA hMSCs. MSCs of OA patients were isolated from aspirates from the intramedullary canal of donors (50-80 years of age) undergoing hip replacement surgery for OA and were treated with or without Naproxen (100 µg/mL). Protein expression and phosphorylation were determined by immunoblotting using specific antibodies (COL X, p38 mitogen-activated protein kinase [p38], phosphorylated-p38, c-Jun N-terminal kinase [JNK], phosphorylated-JNK, extracellular signal-regulated kinase [ERK], and phosphorylated-ERK). Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the expression of COL10A1 and Runt-related transcription factor 2 gene (Runx2). Our results show that Naproxen significantly stimulated COL X protein expression after 72 h of exposure both in normal and OA hMSCs. The basal phosphorylation of mitogen-activated protein kinases (MAPKs) (ERK, JNK, and p38) in OA hMSCs was significantly higher than in normal. Naproxen significantly increased the MAPK phosphorylation in normal and OA hMSCs. NSAID cellular effects include cyclooxygenase, 5-lipoxygenase, and p38 MAPK signaling pathways. To investigate the involvement of these pathways in the Naproxen-induced COL10A1 expression, we incubated normal and OA hMSCs with Naproxen with and without inhibitors of ERK (U0126), JNK (BI-78D3), p38 (SB203580), and 5-lipoxygenase (MK-886). Our results showed that increased basal COL10A1 expression in OA hMSCs was significantly suppressed in the presence of JNK and p38 inhibitors, whereas Naproxen-induced COL10A1 expression was suppressed by 5-lipoxygenase inhibitor. This study shows that Naproxen induces COL X both at transcriptional and translational levels in normal and OA hMSCs. Elevated basal COL10A1 expression in OA hMSCs is probably through the activation of MAPK pathway and Naproxen-induced COL10A1 expression is through the increased 5-lipoxygenase signaling.