Polymyalgia rheumatica: inflammation suppression with low dose of methylprednisolone or modified-release prednisone.

OBJECTIVE: Polymyalgia rheumatica (PMR) is an inflammatory disease that affects people aged > 50 years, and is characterised by pain and morning stiffness in the shoulder and pelvic girdle with synovitis of the proximal joints and extra-articular synovial structures. It is currently mainly treated with glucocorticoids (GCs). The aim of the study was to evaluate changes in inflammatory markers and their correlations with cortisol levels after treatment with 6-methylprednisolone (6-MP) or modified-release prednisone (MR-P) in patients with "early" PMR. PATIENTS AND METHODS: The study involved 81 GC-naïve with "early" PMR diagnosed on the basis of the 2012 EULAR/ACR criteria: 38 treated with 6-MP at a starting dose of 12 mg at 8.00 a.m, gradually tapered to 8, 4 and 2 mg/day, and 43 treated with MR-P at a starting dose of 10 mg at 10 p.m, tapered to 7, 5, 3, 2 and 1 mg. The markers of inflammation (ESR mm/h, CRP mg/dL and fibrinogen mg/dL), the circulating serum levels of cytokines (TNFa and IL-6), and morning serum cortisol levels were evaluated at baseline and during GC treatment. RESULTS: There were significant differences between baseline and the end of treatment in the serum levels of IL-6 (5.3 ± 9.3 vs 2.8 ± 3.3 pg/mL; p < 0.05) and CRP (2.1 ± 3.3 vs 0.9 ± 1.7 mg/dL; p < 0.01) in the patients treated with MR-P, and in serum cortisol levels (15.8±6.4 vs 13.6+5.6 µg/dL; p < 0.01) in the patients treated with 6-MP. After the first month of treatment, 76.7% of the patients treated with MR-P had IL6 levels at or below the upper normal limit, whereas 52.6% of those treated with 6-MP had normal IL6 levels (p < 0.05). There was also a significant difference in the percentage of patients in whom the daily GC dose was tapered within eight months (6.7% in the MR-P group vs 25% in the 6-MP group; p < 0.001) and, by the end of the study, respectively 59.5% vs 35.1% patients were receiving a low GC dose or had discontinued treatment altogether (OR 2.7, 95% CI 1.0-6.77; p < 0.001). After six and 12 months, respectively 10.3% and 14.3% of the patients had discontinued MR-P, as against none of the patients treated with 6-MP (p < 0.05). CONCLUSIONS: In this prospective observational study of PMR patients receiving low-dose GCs, the changes in inflammatory markers were similar in those treated with 6-MP or MR-P, whereas morning cortisol levels remained unchanged only in the MR-P group. During the first month of treatment, MR-P chronotherapy given at bedtime significantly decreased IL-6 levels. The percentage of patients stopping GC treatment was higher in the MR-P group than in the 6-MP group.

Characterization and sorting of flow cytometric populations in human semen.

Human semen is a complex biological matrix. It contains mature spermatozoa, immature germ cells, residual apoptotic bodies and, in some cases, epithelial cells and leucocytes. Hence, one of the challenges in applying flow cytometry in spermatology is the correct recognition of spermatozoa and their separation from signals of other semen cells/elements. In this study, we show that semen spermatozoa are included in a well-defined, flame-shaped FSC/SSC region (FR), by demonstrating that the count of the spermatozoa contained in such region overlaps that obtained by microscopy in the same samples. In FR, nuclear staining of semen samples reveals three different populations: unstained, brighter and dimmer. Unstained elements were previously characterized as apoptotic bodies of testis origin and the brighter elements represent the majority of semen spermatozoa, whereas the composition and the origin of the population with a lower nuclear staining is less clear, albeit we have previously shown that all the elements constituting it are positive for TUNEL. In this study, we sorted all the elements contained in FR region and demonstrated that the dimmer elements are spermatozoa. To further characterize dimmer spermatozoa, we evaluated apoptotic caspases and chromatin immaturity, the latter detected by aniline blue (AB) and chromomycin A (CMA3) staining. We found that caspases were much more expressed in the dimmer spermatozoa (71.4 ± 18.8%) than in the brighter (46.7 ± 15.1%), whereas similar amounts of spermatozoa with chromatin immaturity were found in both populations (brighter, AB: 48.2 ± 19.5%; CMA3: 48.5 ± 20.4% and dimmer, AB: 43.4 ± 19.8%; CMA3: 36.1 ± 18.0%). Hence, the role of apoptosis in generating dimmer spermatozoa and their DNA fragmentation appears clear, whereas the involvement of defects during the chromatin packaging remains elusive.

Highlights on novel technologies for the detection of antibodies to Ro60, Ro52, and SS-B.

OBJECTIVE: We aimed to compare a chemiluminescent immunoassay (CIA, QUANTA Flash) on BIO-FLASH with a multiplex flow immunoassay (MFI) on BioPlex 2200 for the detection of antibodies to Ro60, Ro52, and SS-B. METHODS: The study included 241 samples, from patients suffering from systemic autoimmune diseases (n = 108) as well as disease controls (n = 133). All samples were tested for anti-Ro52, anti-Ro60, and anti-SS-B (La) antibodies on QUANTA Flash (INOVA Diagnostics, San Diego, USA) and BioPlex 2200 (Bio-Rad Laboratories Inc., Hercules, USA). Discrepant samples were tested by two independent methods: BlueDot/ANA and QUANTRIX Microarray (both D-tek, Belgium). RESULTS: The overall qualitative agreements were 95.4% (95% confidence interval, CI 92.0-97.7%) for anti-Ro52, 98.8% (95% CI 96.4-99.7%) for anti-Ro60, and 91.7% (95% CI 87.5-94.9%) for anti-SS-B antibodies. There were 34 discrepant samples among all assays (20 anti-SS-B, 11 anti-Ro52, 3 anti-Ro60). 30/33 of retested samples (by D-tek dot blot) agreed with the QUANTA Flash results. Similar findings were obtained with QUANTRIX Microarray kit. CONCLUSION: QUANTA Flash and BioPlex 2200 show good qualitative agreement. The clinical performances were similar for anti-Ro52 and anti-Ro60 autoantibodies while differences were observed for anti-SS-B (La) antibodies.

Development of a specific method to evaluate 8-hydroxy, 2-deoxyguanosine in sperm nuclei: relationship with semen quality in a cohort of 94 subjects.

Oxidative stress (OS) is involved in many disoders including male infertility. Human spermatozoa are very sensitive targets of reactive oxygen species (ROS) and most sperm functions are impaired in the case of OS. In addition unbalanced production of ROS is considered one of the most important causes of sperm DNA fragmentation, a semen trait of infertile men. The relationship between oxidative damage and semen quality is partially controversial, probably due to the different methods and/or targets used to reveal the OS. In this study, by fluorescence microscopy and flow cytometry, we compared two methods to reveal 8-hydroxy,2-deoxyguanosine (8-OHdG), the hallmark of oxidative DNA damage: an immunofluorescence method and the commercial OxyDNA kit. We found that although both methods localized the labelling in sperm nuclei they yielded different measures, and only with the immunofluorescence method was the labelling specific for sperm 8-OHdG. The immunofluorescence method, coupled to flow cytometry, was thus selected to analyse the 8-OHdG content in semen samples from 94 subfertile patients and to investigate the relationship with semen quality. We found that the percentages of spermatozoa with 8-OHdG (mean±s.d., 11.4±6.9%) were related to sperm count (Pearson's correlation coefficient (r)=-0.27, P=0.04 (ANOVA and student's t-test)), motility (progressive: r=-0.22, P=0.04; non-progressive: r=0.25, P=0.01), and normal morphology (r=-0.27, P=0.01). In conclusion, we demonstrate that immunofluorescence/flow cytometry is a reliable and specific method to detect 8-OHdG at single-cell level and show that oxidative damage only partially overlaps poor semen quality, suggesting that it could provide additional information on male fertility with respect to routine semen analysis.

WIP1 phosphatase modulates the Hedgehog signaling by enhancing GLI1 function.

The Hedgehog-GLI (HH-GLI) signaling plays a critical role in controlling growth and tissue patterning during embryogenesis and is implicated in a variety of human malignancies, including those of the skin. Phosphorylation events have been shown to regulate the activity of the GLI transcription factors, the final effectors of the HH-GLI signaling pathway. Here, we show that WIP1 (or PPM1D), an oncogenic phosphatase amplified/overexpressed in several types of human cancer, is a positive modulator of the HH signaling. Mechanistically, WIP1 enhances the function of GLI1 by increasing its transcriptional activity, nuclear localization and protein stability, but not of GLI2 nor GLI3. We also find that WIP1 and GLI1 are in a complex. Modulation of the transcriptional activity of GLI1 by WIP1 depends on the latter's phosphatase activity and, remarkably, does not require p53, a known WIP1 target. Functionally, we find that WIP1 is required for melanoma and breast cancer cell proliferation and self-renewal in vitro and melanoma xenograft growth induced by activation of the HH signaling. Pharmacological blockade of the HH pathway with the SMOOTHENED antagonist cyclopamine acts synergistically with inhibition of WIP1 in reducing growth of melanoma and breast cancer cells in vitro. Overall, our data uncover a role for WIP1 in modulating the activity of GLI1 and in sustaining cancer cell growth and cancer stem cell self-renewal induced by activation of the HH pathway. These findings open a novel therapeutic approach for human melanomas and, possibly, other cancer types expressing WIP1 and with activated HH pathway.