Biagio Olivito

Th17 plasticity in human autoimmune arthritis is driven by the inflammatory environment

In several murine models of autoimmune arthritis, Th17 cells are the dominant initiators of inflammation. In human arthritis the majority of IL-17–secreting cells within the joint express a cytokine phenotype intermediate between Th17 and Th1. Here we show that Th17/1 cells from the joints of children with inflammatory arthritis express high levels of both Th17 and Th1 lineage-specific transcription factors, RORC2 and T-bet. Modeling the generation of Th17/1 in vitro, we show that Th17 cells “convert” to Th17/1 under conditions that mimic the disease site, namely low TGFβ and high IL-12 levels, whereas Th1 cells cannot convert to Th17. Th17/1 cells from the inflamed joint share T-cell receptor (TCR) clonality with Th17 cells, suggesting a shared clonal origin between Th17 and Th17/1 cells in arthritis. Using CD161, a lectin-like receptor that is a marker of human Th17, we show synovial Th17 and Th17/1 cells, and unexpectedly, a large proportion of Th1 cells express CD161. We provide evidence to support a Th17 origin for Th1 cells expressing CD161. In vitro, Th17 cells that convert to a Th1 phenotype maintain CD161 expression. In the joint CD161+ Th1 cells share features with Th17 cells, with shared TCR clonality, expression of RORC2 and CCR6 and response to IL-23, although they are IL-17 negative. We propose that the Th17 phenotype may be unstable and that Th17 cells may convert to Th17/1 and Th1 cells in human arthritis. Therefore therapies targeting the induction of Th17 cells could also attenuate Th17/1 and Th1 effector populations within the inflamed joint.

HEDGEHOG‐GLI Signaling Drives Self‐Renewal and Tumorigenicity of Human Melanoma‐Initiating Cells

The question of whether cancer stem/tumor‐initiating cells (CSC/TIC) exist in human melanomas has arisen in the last few years. Here, we have used nonadherent spheres and the aldehyde dehydrogenase (ALDH) enzymatic activity to enrich for CSC/TIC in a collection of human melanomas obtained from a broad spectrum of sites and stages. We find that melanomaspheres display extensive in vitro self‐renewal ability and sustain tumor growth in vivo, generating human melanoma xenografts that recapitulate the phenotypic composition of the parental tumor. Melanomaspheres express high levels of Hedgehog (HH) pathway components and of embryonic pluripotent stem cell factors SOX2, NANOG, OCT4, and KLF4. We show that human melanomas contain a subset of cells expressing high ALDH activity (ALDHhigh), which is endowed with higher self‐renewal and tumorigenic abilities than the ALDHlow population. A good correlation between the number of ALDHhigh cells and sphere formation efficiency was observed. Notably, both pharmacological inhibition of HH signaling by the SMOOTHENED (SMO) antagonist cyclopamine and GLI antagonist GANT61 and stable expression of shRNA targeting either SMO or GLI1 result in a significant decrease in melanoma stem cell self‐renewal in vitro and a reduction in the number of ALDHhigh melanoma stem cells. Finally, we show that interference with the HH‐GLI pathway through lentiviral‐mediated silencing of SMO and GLI1 drastically diminishes tumor initiation of ALDHhigh melanoma stem cells. In conclusion, our data indicate an essential role of the HH‐GLI1 signaling in controlling self‐renewal and tumor initiation of melanoma CSC/TIC. Targeting HH‐GLI1 is thus predicted to reduce the melanoma stem cell compartment. Stem Cells2012;30:1808–1818

Investigation on the Origin of Sperm DNA Fragmentation: Role of Apoptosis, Immaturity and Oxidative Stress.

Sperm DNA fragmentation (sDF) represents a threat to male fertility, human reproduction and the health of the offspring. The causes of sDF are still unclear, even if apoptosis, oxidative assault and defects in chromatin maturation are hypothesized. Using multicolor flow cytometry and sperm sorting, we challenged the three hypothesized mechanisms by simultaneously evaluating sDF and signs of oxidative damage (8-hydroxy, 2′-deoxyguanosine (8-OHdG) and malondialdehyde (MDA)), apoptosis (caspase activity and cleaved poly(ADP-ribose) polymerase (cPARP)) and sperm immaturity (creatine phosphokinase (CK) and excess of residual histones). Active caspases and c-PARP were concomitant with sDF in a high percentage of spermatozoa (82.6% ± 9.1% and 53.5% ± 16.4%, respectively). Excess of residual histones was significantly higher in DNA-fragmented sperm versus sperm without DNA fragmentation (74.8% ± 17.5% and 37.3% ± 16.6%, respectively, p < 0.005), and largely concomitant with active caspases. Conversely, oxidative damage was scarcely concomitant with sDF in the total sperm population, at variance with live sperm, where 8-OHdG and MDA were clearly associated to sDF. In addition, most live cells with active caspase also showed 8-OHdG, suggesting activation of apoptotic pathways in oxidative-injured live cells. This is the first investigation on the origin of sDF directly evaluating the simultaneous presence of the signs of the hypothesized mechanisms with DNA breaks at the single cell level. The results indicate that the main pathway leading to sperm DNA breaks is a process of apoptosis, likely triggered by an impairment of chromatin maturation in the testis and by oxidative stress during the transit in the male genital tract. These findings are highly relevant for clinical studies on the effects of drugs on sDF and oxidative stress in infertile men and for the development of new therapeutic strategies.

Th17 Transcription Factor RORC2 Is Inversely Correlated with FOXP3 Expression in the Joints of Children with Juvenile Idiopathic Arthritis

Objective. To investigate the relationship between interleukin 17 (IL-17) producing T cells (Th17) and CD4+CD25+FOXP3+ regulatory T cells (Tregs) in blood and synovial fluid (SF) of patients with juvenile idiopathic arthritis (JIA). Methods. Sixty-five children with JIA (18 males and 47 females, median age 6.2 yrs; 45 with oligoarticular and 20 with polyarticular course) and 75 age- and sex-matched healthy controls were studied. Flow cytometry was used to analyze the forkhead box P3 (FOXP3)-positive Treg cells in peripheral blood (PB) and synovial fluid mononuclear cells (SFMC). FOXP3 and retinoic-acid related orphan receptor C isoform 2 (RORC2) messenger RNA (mRNA) were assessed by real-time polymerase chain reaction analysis. Cytokines (IL-17 and Th1/Th2 related cytokines) were measured in culture supernatants of 11 paired PBMC and SFMC activated with PMA and ionomycin. Results. FOXP3+ T cells and FOXP3 mRNA amounts were significantly lower in PB of children with JIA as compared with controls (p = 0.0002 and p = 0.001, respectively) and a higher percentage of Treg cells with concomitant higher level of FOXP3 transcript levels were observed in SF when compared with their PB counterparts (both p < 0.0001). SF CD4+FOXP3+ T cells were characterized by higher amounts of FOXP3 protein per cell when compared with peripheral CD4+FOXP3+ T cells, as revealed by the difference in FOXP3 median fluorescence intensity (median ± SD, arbitrary units, 54 ± 22.6 vs 19.5 ± 4.2; p < 0.001). RORC2 transcript levels were higher in JIA joints when compared with matched PB samples (median fold increase 3.9, p < 0.0001) but negatively correlated with FOXP3 mRNA levels (r = −0.623, p = 0.04). Stimulated SFMC displayed an impaired ability to produce IL-17 when compared with PBMC and, interestingly, an inverse relationship between IL-17 levels and the percentage of CD4+CD25+FOXP3+ SF T cells (r = −0.510, p = 0.047) was seen. Conclusion. We demonstrated for the first time an increased synovial expression of the transcription factor of Th17, RORC2, in JIA, and its inverse relationship with FOXP3 mRNA. These results extend research on “Th17” and Tregs in JIA.

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.

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 (Pearsons correlation coefficient (r)=-0.27, P=0.04 (ANOVA and students 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.

Interferon-gamma release assay sensitivity in children younger than 5 years is insufficient to replace the use of tuberculin skin test in western countries.

Tuberculin skin test, QuantiFERON-TB Gold In-Tube and T-SPOT.TB were performed in 338 children at risk for tuberculosis (TB), including 70 active TB cases. In children <5 years of age, QuantiFERON-TB Gold In-Tube sensitivity was 73.3% [95% confidence interval (CI): 57.5–89.1]; and T-SPOT.TB sensitivity was 59.3% (95% CI: 40.1–77.8); both were inferior to tuberculin skin test sensitivity (90.0%; 95% CI: 79.3–100). In children ≥5 years QuantiFERON-TB Gold In-Tube sensitivity was 92.5% (95% CI: 84.4–100); T-SPOT.TB sensitivity was 73.0% (95% CI: 58.6–87.3) ; and tuberculin skin test sensitivity was 97.5% (95% CI: 92.6–100).Test specificities were similar in all age groups.

Autoimmunity and Extrahepatic Manifestations in Treatment-Naïve Children with Chronic Hepatitis C Virus Infection

Hepatitis C virus (HCV) infection has been associated with autoimmunity and extrahepatic manifestations in adults. Few data are available on these topics in children. Nonorgan specific auto-antibodies development is part of the natural course of chronic hepatitis C in children. Smooth muscle autoantibody is the most common autoantibody found, while liver-kidney microsomal type-1 antibody positivity is the most peculiar autoimmune feature of children with HCV infection. The clinical significance of non-organ specific autoantibodies in the course of paediatric chronic hepatitis C is still debated. Autoantibody positivity can be considered neutral for most patients, while it can be associated with negative connotations for others, especially those positive for liver-kidney microsomal type-1 autoantibody. Subclinical hypothyroidism but not autoimmune thyroiditis has been demonstrated in HCV infection in children, while only few cases of HCV-associated membranoproliferative glomerulonephritis have been described. Single reports are available in the literature reporting the anecdotal association between chronic hepatitis C and other extrahepatic manifestations such as myopathy and opsoclonus-myoclonus syndrome. Despite the low incidence of extrahepatic manifestations of chronic hepatitis C in children, overall, available data suggest a careful monitoring.

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.

FOXP3 expression in peripheral blood and synovial cells of patients with juvenile idiopathic arthritis: relationship with IL-17 at cytokine and molecular level

Materials and methods We have enrolled 58 JIA patients (polyarticular and oligoarticular disease) and 69 healthy controls. We examined CD4+CD25+FOXP3+ percentage (flow cytometry), FOXP3 and RORγt mRNA (RT-PCR) in peripheral blood mononuclear cells (PBMCs), and in synovial fluid mononuclear cells (SFMCs). FOXP3 median fluorescence intensity (MFI) of CD4+ T cells was also determined. Interleukin-17 levels were measured (ELISA) in stimulated PBMCs supernatants in 22 patients.