Intra and extracellular functions of sphingosine-1-phosphate in sterile inflammation.

Sterile inflammation is a key component of a variety of diseases including, gout, arthritis, type 1 diabetes, Alzheimer’s disease and multiple sclerosis (MS). Sterile inflammation induces the recruitment of immune cells via chemokines, such as CCL5 and CXCL10. Expression of these chemokines is dependent on IRF-1. Recently the FDA has approved the use of a pro-drug, FTY720 that after phosphorylation becomes a S1P mimetic for the treatment of MS. This report describes two novel and opposing mechanisms of S1P action in sterile inflammation. First, intracellular S1P acts as a cofactor of cIAP2 that inducesIL-1-dependent K63-polyubiquitination of IRF-1, which leads to the recruitment of immune cells to the site of inflammation. Conversely, extracellular S1P provides a feedback loop that inhibits CXCL10 and CCL5 expression through S1PR2 signaling. Accordingly, immune cell infiltration to sites of sterile inflammation is increased in S1PR2-/- production via calcium-dependent, but cAMP- and PKA-independent mechanisms that likely involve c-Fos expression and unconventional PKC activation. Elevated c-Fos could competitively inhibit CCL5 expression directly or indirectly via blocking IFN production. These two novel pathways highlight unexpected aspects of S1P signaling, and provide potential mechanisms that can be exploited for the improvement of therapeutics for the treatment of MS

¹⁵N-thymdine administration and quantification in urine in infants.

(a) Tetralogy of Fallot (ToF) infants were given 15N-thymidine at home with the exception of one patient and heart failure (HF) infants received 15N-thymidine at the hospital. Each infant received five oral doses of 50 mg/kg 15N-thymidine in individual single-dose syringes over the course of five consecutive days. Families filled out daily logs to record timing of doses, urine output, and any issues with dose administration. Cotton balls were placed in diapers and collected in specimen bags after infants voided. Cotton balls were collected up to 24 hours following the last dose. Cotton balls that dried during transportation and handling processes were discarded. Viable cotton balls were squeezed to express urine samples into separate 1.5 mL Eppendorf tubes and urine volumes were recorded. Samples were dried, crushed using forceps and weighed. Dried samples were analyzed by elemental analyzer isotope ratio mass spectrometry (IRMS). Atomic ratios of 15N/14N in each urine sample were expressed. (b) Enrollment and follow up of all study participants.</p

Comparing different fitting functions for aggregate data of ¹⁵N urine enrichment from infants with ToF or HF.

Comparing different fitting functions for aggregate data of 15N urine enrichment from infants with ToF or HF.</p

¹⁵N enrichment levels after ¹⁵N-thymidine administration show temporal patterns in infants with tetralogy of Fallot (ToF).

Analysis of urine samples from six infants with ToF/PS who had received oral 15N-thymidine (50 mg/kg/day) was performed. Each graph has the patient study identifier and age at 15N-thymidine labeling indicated. Urine samples were extracted from diapers collected at home, dried, and subjected to Isotope Ratio Mass Spectrometry (IRMS) to quantify 15N/14N atomic ratios. The x-axes indicate timings of 15N-thymidine doses beginning with 00:00 hour of the day, and the Y-axes indicate the 15N/14N atomic ratios. Timing of 15N-thymidine administration is indicated with vertical green arrows (↓) and dotted lines. Each symbol (●) indicates one urine sample. Results of single samples are connected by solid black lines, unless > 24 hr apart. The threshold, i.e., the physiological 15N/14N atomic ratio, is indicated with a dotted red horizontal line.</p

Primary data for examining appropriateness of third-degree polynomial fitting.

Primary data for examining appropriateness of third-degree polynomial fitting.</p

IRB approval summary for observational study.

BackgroundWe have developed a new clinical research approach for the quantification of cellular proliferation in human infants to address unanswered questions about tissue renewal and regeneration. The approach consists of oral 15N-thymidine administration to label cells in S-phase, followed by Multi-isotope Imaging Mass Spectrometry for detection of the incorporated label in cell nuclei. To establish the approach, we performed an observational study to examine uptake and elimination of 15N-thymidine. We compared at-home label administration with in-hospital administration in infants with tetralogy of Fallot, a form of congenital heart disease, and infants with heart failure.MethodsWe examined urine samples from 18 infants who received 15N-thymidine (50 mg/kg body weight) by mouth for five consecutive days. We used Isotope Ratio Mass Spectrometry to determine enrichment of 15N relative to 14N (%) in urine.Results/findings15N-thymidine dose administration produced periodic rises of 15N enrichment in urine. Infants with tetralogy of Fallot had a 3.2-fold increase and infants with heart failure had a 4.3-fold increase in mean peak 15N enrichment over baseline. The mean 15N enrichment was not statistically different between the two patient populations (p = 0.103). The time to peak 15N enrichment in tetralogy of Fallot infants was 6.3 ± 1 hr and in infants with heart failure 7.5 ± 2 hr (mean ± SEM). The duration of significant 15N enrichment after a dose was 18.5 ± 1.7 hr in tetralogy of Fallot and in heart failure 18.2 ± 1.8 hr (mean ± SEM). The time to peak enrichment and duration of enrichment were also not statistically different (p = 0.617 and p = 0.887).ConclusionsThe presented results support two conclusions of significance for future applications: (1) Demonstration that 15N-thymidine label administration at home is equivalent to in-hospital administration. (2) Two different types of heart disease show no differences in 15N-thymidine absorption and elimination. This enables the comparative analysis of cellular proliferation between different types of heart disease.</div

Recruitment and inclusion of results from 18 infants with heart disease.

(a) Consort diagram shows that 13 infants with tetralogy of Fallot (ToF) were enrolled and received label; data from 6 infants was analyzed. (b) Consort diagram shows that 5 infants with heart failure (HF) were enrolled, received label, and urine was collected for analysis.</p

Summary of comparison between ToF and HF infants.

BackgroundWe have developed a new clinical research approach for the quantification of cellular proliferation in human infants to address unanswered questions about tissue renewal and regeneration. The approach consists of oral 15N-thymidine administration to label cells in S-phase, followed by Multi-isotope Imaging Mass Spectrometry for detection of the incorporated label in cell nuclei. To establish the approach, we performed an observational study to examine uptake and elimination of 15N-thymidine. We compared at-home label administration with in-hospital administration in infants with tetralogy of Fallot, a form of congenital heart disease, and infants with heart failure.MethodsWe examined urine samples from 18 infants who received 15N-thymidine (50 mg/kg body weight) by mouth for five consecutive days. We used Isotope Ratio Mass Spectrometry to determine enrichment of 15N relative to 14N (%) in urine.Results/findings15N-thymidine dose administration produced periodic rises of 15N enrichment in urine. Infants with tetralogy of Fallot had a 3.2-fold increase and infants with heart failure had a 4.3-fold increase in mean peak 15N enrichment over baseline. The mean 15N enrichment was not statistically different between the two patient populations (p = 0.103). The time to peak 15N enrichment in tetralogy of Fallot infants was 6.3 ± 1 hr and in infants with heart failure 7.5 ± 2 hr (mean ± SEM). The duration of significant 15N enrichment after a dose was 18.5 ± 1.7 hr in tetralogy of Fallot and in heart failure 18.2 ± 1.8 hr (mean ± SEM). The time to peak enrichment and duration of enrichment were also not statistically different (p = 0.617 and p = 0.887).ConclusionsThe presented results support two conclusions of significance for future applications: (1) Demonstration that 15N-thymidine label administration at home is equivalent to in-hospital administration. (2) Two different types of heart disease show no differences in 15N-thymidine absorption and elimination. This enables the comparative analysis of cellular proliferation between different types of heart disease.</div

Recruited patients with severe HF on the wait list for heart transplantation.

Recruited patients with severe HF on the wait list for heart transplantation.</p