Fingolimod therapy in multiple sclerosis leads to the enrichment of a subpopulation of aged NK cells

Fingolimod is an approved oral treatment for relapsing–remitting multiple sclerosis (RRMS) that modulates agonistically the sphingosin-1-phosphate receptor (S1PR), inhibiting thereby the egress of lymphocytes from the lymph nodes. In this interventional prospective clinical phase IV trial, we longitudinally investigated the impact of fingolimod on frequencies of NK cell subpopulations by flow cytometry in 17 RRMS patients at baseline and 1, 3, 6, and 12 months after treatment initiation. Clinical outcome was assessed by the Expanded Disability Status Scale (EDSS) and annualized relapse rates (ARR). Over the study period, median EDSS remained stable from month 3 to month 12, and ARR decreased compared to ARR in the 24 months prior treatment. Treatment was paralleled by an increased frequency of circulating NK cells, due primarily to an increase in CD56(dim)CD94(low) mature NK cells, while the CD56(bright) fraction and CD127(+) innate lymphoid cells (ILCs) decreased over time. An unsupervised clustering algorithm further revealed that a particular fraction of NK cells defined by the expression of CD56(dim)CD16(++)KIR(+/-)NKG2A(-)CD94(-)CCR7(+/-)CX(3)CR1(+/-)NKG2C(-)NKG2D(+)NKp46(-)DNAM1(++)CD127(+) increased during treatment. This specific phenotype might reflect a status of aged, fully differentiated, and less functional NK cells. Our study confirms that fingolimod treatment affects both NK cells and ILC. In addition, our study suggests that treatment leads to the enrichment of a specific NK cell subset characterized by an aged phenotype. This might limit the anti-microbial and anti-tumour NK cell activity in fingolimod-treated patients

Impaired response of blood neutrophils to cell-death stimulus differentiates AQP4-IgG-seropositive NMOSD from MOGAD

BACKGROUND: In neuromyelitis optica spectrum disorders (NMOSD) and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), neutrophils are found in CNS lesions. We previously demonstrated that NMOSD neutrophils show functional deficiencies. Thus, we hypothesized that neutrophil accumulation in the CNS may be facilitated by impairments affecting mechanisms of neutrophil death. OBJECTIVE: To evaluate cell death in blood neutrophils from aquaporin-4 (AQP4)-IgG-seropositive NMOSD and MOGAD patients as well as matched healthy controls (HC) using in vitro assays. METHODS: Twenty-eight AQP4 + NMOSD and 19 MOGAD patients in stable disease phase as well as 45 age- and sex-matched HC were prospectively recruited. To induce cell death, isolated neutrophils were cultured with/without phorbol 12-myristate 13-acetate (PMA). Spontaneous and PMA-induced NETosis and apoptosis were analyzed using 7-AAD and annexin-V by flow cytometry. Caspase-3 was assessed by western blot. Myeloperoxidase-DNA complexes (MPO-DNA), MPO and elastase were evaluated by ELISA, and cell-free DNA (cfDNA) by a fluorescence-based assay. Reactive oxygen species (ROS) were evaluated by a dihydrorhodamine 123-based cytometric assay. Serum GM-CSF, IL-6, IL-8, IL-15, TNF-ɑ and IL-10 were evaluated by multiplex assays, and neurofilament light chain (NfL) by single-molecule array assay. RESULTS: In response to PMA, neutrophils from AQP4 + NMOSD but not from MOGAD patients showed an increased survival, and subsequent reduced cell death (29.6% annexin V(+) 7-AAD(+)) when compared to HC (44.7%, p = 0.0006). However, AQP4 + NMOSD also showed a mild increase in annexin V(+) 7-AAD(-) early apoptotic neutrophils (24.5%) compared to HC (20.8%, p = 0.048). PMA-induced reduction of caspase-3 activation was more pronounced in HC (p = 0.020) than in AQP4 + NMOSD neutrophils (p = 0.052). No differences were observed in neutrophil-derived MPO-DNA or serum levels of MPO, elastase, IL-6, IL-8 and TNF-ɑ. IL-15 levels were increased in both groups of patients. In AQP4 + NMOSD, an increase in cfDNA, GM-CSF and IL-10 was found in serum. A positive correlation among cfDNA and NfL was found in AQP4 + NMOSD. CONCLUSIONS: AQP4 + NMOSD neutrophils showed an increased survival capacity in response to PMA when compared to matched HC neutrophils. Although the data indicate that the apoptotic but not the NETotic response is altered in these neutrophils, additional evaluations are required to validate this observation

The effects of a loading dose followed by constant rate infusion of xylazine compared with romifidine on sedation, ataxia and response to stimuli in horses

Objective: To compare xylazine and romifidine constant rate infusion (CRI) protocols regarding degree of sedation, and effects on postural instability (PI), ataxia during motion (A) and reaction to different stimuli. Study design: Blinded randomized experimental cross-over study. Animals: Ten adult horses. Methods: Degree of sedation was assessed by head height above ground (HHAG). Effects on PI, A and reaction to visual, tactile and acoustic stimulation were assessed by numerical rating scale (NRS) and by visual analogue scale (VAS). After baseline measurements, horses were sedated by intravenous loading doses of xylazine (1 mg kg−1) or romifidine (80 μg kg−1) administered over 3 minutes, immediately followed by a CRI of xylazine (0.69 mg kg−1 hour−1) or romifidine (30 μg kg−1 hour−1) which was administered for 120 minutes. Degree of sedation, PI, A and reaction to the different stimuli were measured at different time points before, during and for one hour after discontinuing drug administration. Data were analysed using two-way repeated measures anova, a Generalized Linear Model and a Wilcoxon Signed Rank Test (p < 0.05). Results: Significant changes over time were seen for all variables. With xylazine HHAG was significantly lower 10 minutes after the loading dose, and higher at 150 and 180 minutes (i.e. after CRI cessation) compared to romifidine. Reaction to acoustic stimulation was significantly more pronounced with xylazine. Reaction to visual stimulation was greater with xylazine at 145 and 175 minutes. PI was consistently but not significantly greater with xylazine during the first 30 minutes. Reaction to touch and A did not differ between treatments. Compared to romifidine, horses were more responsive to metallic noise with xylazine. Conclusions: Time to maximal sedation and to recovery were longer with romifidine than with xylazine. Clinical relevance: With romifidine sufficient time should be allowed for complete sedation before manipulation

Bevacizumab Eligibility in Patients with Metastatic and Recurrent Cervical Cancer: A Retrospective Review

Objective: Bevacizumab is approved for use in combination with chemotherapy for metastatic/recurrent cervical cancer (CC), with increased survival/response rates. However, use of bevacizumab is not always feasible or safe. The purpose of this study was to identify the percentage of metastatic/recurrent CC patients at our institution who would have been eligible to receive bevacizumab. Methods: A retrospective study was conducted to identify metastatic/recurrent CC patients treated at UFHealth between 2006 and 2016. Chart review was performed to determine if the patient met bevacizumab eligibility criteria. Results: In total, 79 patients with metastatic/recurrent CC were identified; 85.5% would have been ineligible to receive bevacizumab, and 14.5% would have been eligible. The most common reason for exclusion was active bleeding (68.4%); 94% of which was vaginal. In all, 27.6% would be excluded due to poor renal function, and 23.7% due to poor performance status (PS). Conclusions: Despite improved survival, only 14.5% of metastatic/recurrent CC patients treated over a 10-year period would have been eligible to receive bevacizumab. Most patients would have been excluded due to active bleeding, most commonly vaginal bleeding, a common complication from their disease. Identifying novel therapies for metastatic/recurrent CC patients with improved safety profiles that would allow for their use in this challenging population is critical