PD1+ T-cells correlate with Nerve Fiber Density as a prognostic biomarker in patients with resected perihilar cholangiocarcinoma.

Background & Aims: Perihilar cholangiocarcinoma (pCCA) is a hepatobiliary malignancy. Nerve fiber invasion (NFI) shows cancer invading the nerve and is considered an aggressive feature. Nerve fiber density (NFD) consists of small nerve fibers without cancer invasion and is divided into high NFD (high numbers of small nerve fibers) or low NFD (low numbers of small nerve fibers). We aim to explore differences in immune cell populations and survival. Approach & Results: We applied multiplex immunofluorescence (mIF) on 47 pCCA surgically resected patients and investigated immune cell composition in the tumor microenvironment (TME) of nerve fiber phenotypes (NFI, high and low NFD). Group comparison was performed and overall survival (OS) was assessed. The NFI Region of Interest (ROI) was measured with highest CD68+ macrophage levels among 3 ROIs (NFI compared to tumor free p= 0.016 and to tumor p=0.034) and PD1 expression on CD8 and were more abundant in the tumor rather than NFI ROI (p= 0.004 and p= 0.0029 respectively). NFD compared to NFI, demonstrated co-expression of CD8+PD1+ as well as CD68+PD1+ to be significantly higher in high NFD patients (p= 0.027 and p= 0.044, respectively). The high NFD OS was 92 months median OS (95% CI:41-142), for low NFD 20 months ((95% CI: 4-36) and for NFI 19 months (95% CI 7-33). High NFD OS was significantly better compared to low NFD (p= 0.046) and NFI (p= 0.032). Conclusions: PD1+ T-cells correlate with high NFD as a prognostic biomarker, the biological pathway behind this needs to be investigated.

Impact of Dose, Sex, and Strain on Oxaliplatin-Induced Peripheral Neuropathy in Mice

Chemotherapy-induced peripheral neuropathy (CIPN) is a common, dose limiting, and long-lasting side effect of chemotherapy treatment. Unfortunately, no treatment has proven efficacious for this side effect. Rodent models play a crucial role in the discovery of new mechanisms underlying the initiation, progression, and recovery of CIPN and the potential discovery of new therapeutics. However, there is limited consistency in the dose, the sex, age, and genetic background of the animal used in these studies and the outcome measures used in evaluation of CIPN rely primarily on noxious and reflexive measures. The main objective of this study was to provide a comprehensive and systematic characterization of oxaliplatin-induced peripheral neuropathy in mice by using a battery of behavioral, sensory, electrophysiological, and morphometric measures in both sexes of the two widely used strains of mice, C57BL/6J and BALB/cJ. Mice received intraperitoneal injections of 3 or 30 mg/kg cumulative doses of oxaliplatin over the course of 2 weeks. Both doses induced long-term and time-dependent mechanical and cold hypersensitivity. Our results show that 30 mg/kg oxaliplatin reduced the locomotor activity in C57BL/6J mice, and C57BL/6J females showed anxiety-like behavior one-week post completion of treatment. In the same dose group, BALB/cJ males and females sustained a larger decrease in sucrose preference than either male or female C57BL/6J mice. Both strains failed to show significant changes in burrowing and nesting behaviors. Two clinically relevant assessments of changes to the peripheral nerve fibers, nerve conduction and intraepidermal nerve fiber density (IENFD) were evaluated. Only BALB/cJ females showed significant reduction in the nerve conduction amplitude 1 week after 30 mg/kg oxaliplatin regimen. Moreover, this dose of the chemo agent reduced the IENF density in both sexes and strains. Our findings suggest that mouse strain, sex, and assay type should be carefully considered when assessing the effects of oxaliplatin and potential therapeutic interventions.

Intraepidermal Nerve fiber Density in healthy Subjects with Diabetic Family History. ¿Is Neuropathy an early Biomarker? A Pilot study

Objective: Evaluate the intraepidermal nerve fiber density in healthy subjects with diabetic family history compared with diabetic patients and controls. Introduction: Neuropathy is the most prevalent chronic complication of diabetes, presenting various symptoms that interfere with daily living activities, psychosocially disability, and reducing life quality. The skin biopsy is recognized as a minimally invasive procedure that allows morphometric quantification of intraepidermal nerve fibers and has made possible the study of peripheral neuropathies involving thin fibers that traditional methods cannot diagnose. Methods: Analytical cross-sectional observational pilot study with seven patients per group including healthy, diabetic, and healthy with diabetic family history subjects. For the statistical analysis, we used the R package, R software version 3.3.2, with a confidence level of 95%. The research was performed with ANOVA and Kruskal-Wallis test to test the primary objective. Results: The density of intraepidermal nerve fibers is similar between the group with diabetic family history 6.8 ± 2.1 (3.5 - 10.1) and diabetic patients 6.3 ± 2.9 (3.5 - 7.05) while the control group reported a density in parameters of normality of 10± 1.2 (8.2 - 10.1) with a p= 0.01 between the three groups. The decrease of intraepidermal nerve fibers showed a tendency to decrease with increasing age and BMI with a ratio coefficient for age of r= -0.342, 95% CI (-0.67 - 0.106), p= 0.129; and for BMI of r= -0.36, 95% CI (-0.685 - 0.0847), p= 0.109. Conclusion: Intraepidermal nerve fiber density is decreased in subjects with a family history of diabetes mellitus type 2 and even more so in diabetics, with no statistical difference.

Optical clearing reveals TNBS-induced morphological changes of VGLUT2-positive nerve fibers in mouse colorectum

Colorectal hypersensitivity and sensitization of both mechanosensitive and mechanically insensitive afferents develop after intracolonic instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS) in the mouse, a model of post-infectious irritable bowel syndrome. In mice in which ~80% of extrinsic colorectal afferents were labeled genetically using the promotor for vesicular glutamate transporter type 2 (VGLUT2), we systematically quantified the morphology of VGLUT2-positive axons in mouse colorectum 7-28 days following intracolonic TNBS treatment. After removal, the colorectum was distended (20 mmHg), fixed with paraformaldehyde, and optically cleared to image VGLUT2-positive axons throughout the colorectal wall thickness. We conducted vector path tracing of individual axons to allow systematic quantification of nerve fiber density and shape. Abundant VGLUT2-positive nerve fibers were present in most layers of the colorectum, except the serosal and longitudinal muscular layers. A small percentage of VGLUT2-positive myenteric plexus neurons was also detected. Intracolonic TNBS treatment significantly reduced the number of VGLUT2-positive nerve fibers in submucosal, myenteric plexus, and mucosal layers at day 7 post TNBS, which mostly recovered by day 28. We also found that almost all fibers in the submucosa were meandering and curvy, with ~10% showing pronounced curviness (quantified by the linearity index). TNBS treatment resulted in a significant reduction of the proportions of pronounced curvy fibers in the rectal region at 28 days post TNBS. Altogether, the present morphological study reveals profound changes in the distribution of VGLUT2-positive fibers in mouse colorectum undergoing TNBS-induced colitis, and draw attention to curvy fibers in the submucosa with potential roles in visceral nociception.

Improvement of Nerve Fiber Density in Fibromyalgia Patients Treated with IVIg

Results: Small fiber neuropathy and fibromyalgia are two conditions that share overlapping features. Although various treatments are available for use in fibromyalgia, the response often remains unsatisfactory. Prior studies have shown that in small fiber neuropathy of autoimmune etiology, intravenous immunoglobulin (IVIg) holds promise as an effective treatment. Methods: Herein we report the use of IVIg in 7 patients who have both fibromyalgia and small fiber neuropathy. Skin punch biopsy evaluating the nerve fiber density was performed prior to diagnosis and after 6 months of IVIg therapy in each individual. Patients’ symptoms were obtained via a fibromyalgia questionnaire pre- and post-treatment. Results and Conclusion: At the end of 6 months therapy, overall patients reported fewer fibromyalgia symptoms and skin biopsy demonstrated improvements as well. This retrospective pilot study suggests IVIg is a viable potential therapy in a subset of fibromyalgia patients who have small fiber neuropathy.

Nerve fibers in the Tumor Microenvironment are co-localized with Tertiary Lymphoid Structures

ABSTRACTBackgroundB cells and tertiary lymphoid structures (TLS) are reported to be important in the improvement of survival of cancer patients. These secondary lymphoid organs have been associated with the generation of an anti-tumor response. Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancer types and the stromal architecture shapes the intratumoral heterogeneity. The stroma of PDAC is a complex system in which crosstalk takes place between cancer-associated fibroblasts, immune cells, endothelial cells and the cancer cells. Besides immune cells and fibroblasts, there is some limited data about the influence of nerve fibers on cancer progression.Patients and methodsNerve Fiber Density (NFD) was analysed in our cohort of 188 patients with Pancreatic Ductal Adenocarcinoma who underwent pancreatic surgery. We used immunohistochemistry and multiplex imaging to phenotype the immune cell infiltrate. The cell detection classifier measured distance from immune cell to cancer gland and with a heat map we could count TLS. By using Machine learning we were able to define the spatial distribution and counting Tertiary Lymphoid Structures.ResultsHigh NFD is significantly associated with prolonged overall survival (HR 1.676 (95%CI 1.126,2.495) for low vs. high NFD, p-value 0.0109). The immune cells surrounding the nerve fibers were phenotyped in B cells, T cells and dendritic follicular cells, matching a TLS. Here we show that small nerve fibers are located at the TLS in Pancreatic Cancer and a high Nerve Fiber Density combined with more than 5 TLS is associated with a better survival (HR 0.388 (95%CI 0.218, 0.689).ConclusionThe co-localization of small nerve fibers with TLS is a new finding which has not been described before. However the precise roles of these TLS and nerve fibers remains unknown. These findings unravel future pathways and has the potential to reach new directions into already existing targeted therapy.

Corneal In Vivo Laser-Scanning Confocal Microscopy Findings in Dry Eye Patients with Sjögren’s Syndrome

Purpose: To evaluate the changes in cornea in Sjögren’s syndrome (SS) with a novel confocal microscopy device. Methods: Twenty-three right eyes of patients with SS (23 women; mean age, 65.4 ± 11.4 years) and 13 right eyes of 13 age- and sex-matched control subjects (13 women; mean age, 68.8 ± 9.8 years) were studied. Furthermore, eight right eyes of patients with SS (8 women; mean age, 66.9 ± 9.6 years) were studied to evaluate the corneal microscopic alterations after the treatment with topical 3% diquafosol sodium eye drops. All cases had tear quantity, tear breakup time (BUT), ocular surface staining measurements, and corneal in vivo laser-scanning confocal microscopy examinations. The density and area of corneal epithelial cells (superficial, wing, and basal), density of corneal stromal cells (anterior, intermediate, and posterior), density and area of corneal endothelial cells, density and morphology of corneal sub-basal nerve plexus, density of corneal sub-basal inflammatory cells were also assessed. Results: The tear quantity, stability, and vital staining scores were significantly worse in patients with SS than in control subjects (p < 0.0001). Corneal superficial epithelial cell density was significantly lower in SS compared with control subjects (p < 0.0001). Corneal superficial epithelial cell area was significantly larger in SS compared with control subjects (p = 0.007). Corneal sub-basal nerve fiber density was lower in SS compared with control subjects (p < 0.0001). Morphological abnormality of nerve fibers was observed in SS patients. Corneal sub-basal inflammatory cell density was significantly higher in SS patients compared with control subjects (p < 0.0001). Furthermore, the mean corneal superficial epithelial cell density and area, inflammatory cell density, corneal sub-basal nerve fiber density, and morphological abnormality of nerve fibers, were improved with topical 3% diquafosol sodium treatment in the dry eye patients with SS (p < 0.05). Conclusions: The diagnostic modality using in vivo laser-scanning confocal microscopy was a useful method for the evaluation of the corneal cell density and area, nerve fiber density and morphology, and inflammatory cell density in patients with SS and also a useful tool in the assessment of treatment effect with topical 3% diquafosol sodium in the SS patients.