Longitudinal analysis of T-cell receptor repertoires reveals persistence of antigen-driven CD4+ and CD8+ T-cell clusters in systemic sclerosis.

The T-cell receptor (TCR) is a highly polymorphic surface receptor that allows T-cells to recognize antigenic peptides presented on the major histocompatibility complex (MHC). Changes in the TCR repertoire have been observed in several autoimmune conditions, and these changes are suggested to predispose autoimmunity. Multiple lines of evidence have implied an important role for T-cells in the pathogenesis of Systemic Sclerosis (SSc), a complex autoimmune disease. One of the major questions regarding the roles of T-cells is whether expansion and activation of T-cells observed in the diseases pathogenesis is antigen driven. To investigate the temporal TCR repertoire dynamics in SSc, we performed high-throughput sequencing of CD4+ and CD8+ TCRß chains on longitudinal samples obtained from four SSc patients collected over a minimum of two years. Repertoire overlap analysis revealed that samples taken from the same individual over time shared a high number of TCRß sequences, indicating a clear temporal persistence of the TCRß repertoire in CD4+ as well as CD8+ T-cells. Moreover, the TCRßs that were found with a high frequency at one time point were also found with a high frequency at the other time points (even after almost four years), showing that frequencies of dominant TCRßs are largely consistent over time. We also show that TCRß generation probability and observed TCR frequency are not related in SSc samples, showing that clonal expansion and persistence of TCRßs is caused by antigenic selection rather than convergent recombination. Moreover, we demonstrate that TCRß diversity is lower in CD4+ and CD8+ T-cells from SSc patients compared with memory T-cells from healthy individuals, as SSc TCRß repertoires are largely dominated by clonally expanded persistent TCRß sequences. Lastly, using "Grouping of Lymphocyte Interactions by Paratope Hotspots" (GLIPH2), we identify clusters of TCRß sequences with homologous sequences that potentially recognize the same antigens and contain TCRßs that are persist in SSc patients. In conclusion, our results show that CD4+ and CD8+ T-cells are highly persistent in SSc patients over time, and this persistence is likely a result from antigenic selection. Moreover, persistent TCRs form high similarity clusters with other (non-)persistent sequences that potentially recognize the same epitopes. These data provide evidence for an antigen driven expansion of CD4+/CD8+ T-cells in SSc.

Fatty Acid and Carnitine Metabolism Are Dysregulated in Systemic Sclerosis Patients.

Systemic sclerosis (SSc) is a rare chronic disease of unknown pathogenesis characterized by fibrosis of the skin and internal organs, vascular alteration, and dysregulation of the immune system. In order to better understand the immune system and its perturbations leading to diseases, the study of the mechanisms regulating cellular metabolism has gained a widespread interest. Here, we have assessed the metabolic status of plasma and dendritic cells (DCs) in patients with SSc. We identified a dysregulated metabolomic signature in carnitine in circulation (plasma) and intracellularly in DCs of SSc patients. In addition, we confirmed carnitine alteration in the circulation of SSc patients in three independent plasma measurements from two different cohorts and identified dysregulation of fatty acids. We hypothesized that fatty acid and carnitine alterations contribute to potentiation of inflammation in SSc. Incubation of healthy and SSc dendritic cells with etoposide, a carnitine transporter inhibitor, inhibited the production of pro-inflammatory cytokines such as IL-6 through inhibition of fatty acid oxidation. These findings shed light on the altered metabolic status of the immune system in SSc patients and opens up for potential novel avenues to reduce inflammation.

Patients with gout have short telomeres compared with healthy participants: association of telomere length with flare frequency and cardiovascular disease in gout.

AIM AND BACKGROUND: Chronic inflammation associates with increased senescence, which is a strong predictor for cardiovascular disease. We hypothesised that inflammation accelerates senescence and thereby enhances the risk of cardiovascular disease in gout. METHODS: We assessed replicative senescence by quantifying telomere length (TL) in a discovery cohort of 145 Dutch patients with gout and 273 healthy individuals and validated our results in 474 patients with gout and 293 healthy participants from New Zealand. Subsequently, we investigated the effect of cardiovascular disease on TL of all participants. Also, we measured TL of CD4+ and CD8+ T lymphocytes, B lymphocytes, monocytes, natural killer cells and plasmacytoid dendritic cells. Additionally, we assessed the potential temporal difference in TL and telomerase activity. RESULTS: TL in PBMCs of healthy donors decreased over time, reflecting normal ageing. Patients with gout demonstrated shorter telomeres (p=0.001, R2=0.01873). In fact, the extent of telomere erosion in patients with gout was higher at any age compared with healthy counterparts at any age (p<0.0001, R2=0.02847). Patients with gout with cardiovascular disease had the shortest telomeres and TL was an independent risk factor for cardiovascular disease in patients with gout (p=0.001). TL was inversely associated with the number of gouty flares (p=0.005). CONCLUSIONS: Patients with gout have shorter telomeres than healthy participants, reflecting increased cellular senescence. Telomere shortening was associated with the number of flares and with cardiovascular disease in people with gout.

Triage of colonoscopies: open access endoscopy versus outpatient consultation with a gastroenterologist.

BACKGROUND AND STUDY AIMS: In many Dutch hospitals, open access referral for colonoscopy is authorized by a gastroenterologist after screening a standard referral letter (SRL) without face-to-face contact with the patient. We investigated the added value of a 7.5 min outpatient consultation with a gastroenterologist (OC), regarding the patient indications, priority for colonoscopy, and the frequency of correct information about patient medications and comorbidities on SRLs. PATIENTS AND METHODS: In a prospective, blinded, single-center study, gastroenterologists assessed SRLs for the accuracy and priority of the colonoscopy request (SRL). These data were compared to results from the OC, and primary outcomes were the number of patients who were not recommended for colonoscopy and priority scheduling of colonoscopy for suspicion of cancer. RESULTS: Patients were analyzed using both SRL and OC and, of 255 patients, 224 of them underwent colonoscopy. Colonoscopy was not recommended for 6.3 % and 11.4 % of patients using the SRL and OC, respectively (P = 0.02). Using the SRL, gastroenterologists did not recommend colonoscopy for seven patients, but the same patients were recommended for colonoscopy when OC was available. This was explained because the indications on the SRL did not match the information obtained from OC. Compared to OC , more colonoscopies were prioritized when the SRL was used to make decisions. Cancer was detected in 7/112 (SRL ) versus 7/65 (OC ) of priority-scheduled patients. SRLs did not report the use of coumarins and insulin in 1.6 % of patients or the prevalence of serious comorbid conditions in 52 % of patients. CONCLUSIONS: A 7.5 min outpatient consultation with a gastroenterologist improved the identification of indications for colonoscopy, decreased priority scheduling of patients, and increased the number of patients diagnosed with cancer in the prioritized group. SRLs frequently omitted patients' medications and comorbidities.

Low serum levels of testosterone in men with minimal traumatic hip fractures.

Sex steroids are essential for accretion and maintenance of bone mass. Their importance for osteoporotic fractures in men, however, are undefined. We determined circulating levels of testosterone (T), non-SHBG-bound T (bT), free testosterone (FT), oestradiol (E2), intact parathormone (iPTH), 25-OH-vitamin D (25(OH)D), and trabecular bone mineral density at spinal level (tBMD) by single quantitative computed tomography (QCT), respectively, in elderly men 1-3.5 months after minimal traumatic hip fractures (MTHF, age=75+/-10 ys, n=27). A group of patients with non-immobilising stroke (S; age=73+/-8 ys, n=12) served as controls. Men with known secondary osteoporosis were excluded from the study. Furthermore, serum levels of T and E2 were compared to healthy controls aged 20-30 years (n=138) and 60-80 years (n=110). In addition a literature-based analysis of studies on testosterone in men hip fractures were conducted. Mean tBMD of men with MTFH (52.7+/-17.6 mg/cm3, T-score=- 4.5+/-0.6) was significantly lower than in men with S (78+/-16.3 mg/cm3, T-score=- 3.5+/-0.8). Significant differences of the means between both groups were observed for T, bT, and FT but not for E2, 25(OH)D, and iPTH, respectively. About 90 % of men with MTHF had T serum levels 2 SD below the mean of young controls. This proportion reduced to 30 % if compared with serum levels of 60-80-year-old healthy men whereas men after S remained well within the normal range adjusted for age. Mean serum levels of iPTH were within the normal range (1-6.8 pmol/l); 25(OH)D serum levels were at the lower end of the normal control levels (30-190 nmol/l). There was an inverse relationship between iPTH and 25(OH)D (r=- 0,4; p<0,03). In conclusion, low serum T is common in men with MTHF and only partly due to age. It appears to be a primary factor in fragility fractures in men and not simply secondary to morbidity following the fracture. In view of the scarce and inconsistent data published on this issue (1 longitudinal and 6 cross-sectional studies) the present study supports the patho-physiological relevance of low serum testosterone for the occurrence of MTHF in men.

Age-related changes of serum sex hormones, insulin-like growth factor-1 and sex-hormone binding globulin levels in men: cross-sectional data from a healthy male cohort.

The age-dependent decline of the gonadal and somatotopic axis has been causally linked to frailty in the elderly by their effects on muscle mass and bone mineral density. However, for healthy men data on serum oestrogens and androgens, as well as IGF-1, as a common outcome measure covering the whole adult age range are scarce. We therefore studied healthy, nonobese male subjects between 20 and 80 years of age to asses their morning concentrations of total (T), free (FT), bioavailable testosterone (bT), oestradiol (E2), bioavailable oestradiol (bE2), oestrone (E1), sex-hormone binding globulin (SHBG), and insulin-like growth factor 1 (IGF-1). Five hundred and seventy-two male healthy volunteers with a BMI < 30 kg/m2 recruited from regular blood donors and senior sports clubs participated in the study. Serum samples were obtained during morning hours and T, FT, E2, E1, SHBG, albumin and IGF-1 were measured by radio-immunoassay systems. In addition, bT and bE2 were calculated. A potential relationship between sex hormones and IGF-1 was tested by multiple regression analysis including age and BMI. Ageing was negatively related to serum levels of sex steroids and IGF-1 (both P < 0.0001) with a mean decrease (youngest vs. oldest) of 51% for T, 64% for FT, 78% for bT, 32% for E2, 62% for bE2, 29% for E1 and 51% for IGF-1 starting in early adulthood whereas SHBG increased after the 5th decade of life (ANOVA P < 0.001). The decline of sex hormones and IGF-1 remained relatively unchanged after adjustment for BMI. Multiple regression analysis revealed an age-and BMI- independent association between oestradiol and IGF-1. In contrast to the female situation sex hormones in healthy, nonobese men decline continuously with age. This process has already started in the third decade, and is paralleled by a decline of IGF-1 serum levels leading to a substantial proportion of elderly men with markedly lowered serum levels of bioavailable sex hormones and IGF-1 compared to the young adult male range. With the recent demonstration of beneficial effects of androgen replacement therapy in healthy males on general well being, muscle mass and bone mineral density the present data may underline the importance of more detailed studies on the biological significance of hormonal changes in men with age.