The association between duration of remission, fatigue, depression and health-related quality of life in Italian patients with systemic lupus erythematosus.

OBJECTIVE: To evaluate the impact of duration of remission on the health-related quality of life (HRQoL) of patients with systemic lupus erythematosus (SLE). METHODS: We conducted a 5-year retrospective study on two Italian cohorts. Remission was defined as a continuative period of no clinical disease activity, according to the Systemic Lupus Erythematosus Disease Activity Index 2 K, and a permitted maximum prednisone dose of 5 mg/day. HRQoL was measured using the 36-Item Short-Form Health Survey (SF36) during the last visit. RESULTS: We enrolled 136 female SLE patients. During observation, 15 (11%) patients had been in remission for ≥1 and <2 years, 15 (11%) for ≥2 and <3 years, 19 (14%) for ≥3 and <4 years, 9 (7%) for ≥4 and <5 years, and 53 (39%) had been in prolonged remission for ≥5 years. In the multivariate model, considering depression and fatigue as covariates, patients in prolonged remission showed significantly better scores in the physical functioning (p = 0.039), role physical (p = 0.029), bodily pain (p = 0.0057), general health (p = 0.0033) and social functioning (p = 0.0085) components of the SF36, compared with those in remission <5 years or unremitted. Subsequent mediation analyses found that these effects were partly influenced by depression. CONCLUSION: Lupus remission could improve the HRQoL of SLE patients, particularly when associated with appropriate management of depression and fatigue.

Health status and concomitant prescription of immunosuppressants are risk factors for hydroxychloroquine non-adherence in systemic lupus patients with prolonged inactive disease.

Background/objective The objectives of this paper are to assess the extent of and the factors associated with hydroxychloroquine (HCQ) non-adherence in systemic lupus erythematosus (SLE) patients with prolonged inactive disease and to investigate relationships between blood HCQ concentration and quality of life (QoL). Methods Consecutive SLE patients, in remission for at least one year and taking a stable dose of HCQ were investigated. At study entry (T0) and six months later (T6) a blood venous sample was taken to measure whole blood concentration of [HCQ] and desethylchloroquine ([DCQ]). Moreover, at T0 each patient completed validated questionnaires assessing QoL, disability, anxiety, depression and visual analogue scales for fatigue, pain, general health (GH), and self-assessment of disease activity. Results Eighty-three patients with a median [HCQ] of 327 ng/ml were enrolled. At T0, 24 (29%) were defined as non-adherent ([HCQ] < 100 ng/ml). At multiple logistic regression analysis the physical summary of SF-36 ( p = 0.038), and the concomitant use of immunosuppressants ( p = 0.010) were independently associated with non-adherence. A significant increase of HCQ adherence was observed at T6 ( p < 0.05). Conclusions A better health status and the concomitant prescription of immunosuppressants represent risk factors for HCQ non-adherence in SLE patients in remission. Monitoring HCQ levels might represent an important opportunity to improve adherence.

Characterization of Follicular Atresia Responsive to BPA in Zebrafish by Morphometric Analysis of Follicular Stage Progression.

Bisphenol A is an industrial chemical compound, pervasively polluting the environment and diet, classified as an endocrine disruptor because of its interference effects on the endocrine system. In zebrafish, BPA exposure induces follicular atresia. To acquire knowledge on this atretic effect, using a qualitative and quantitative histomorphological approach, we studied zebrafish ovarian follicular stage development in response to low BPA concentrations. Results show that BPA interferes with follicular progression by affecting the previtellogenic and vitellogenic phases. In particular, BPA exposure (i) increases follicular recruitment by acting on primary stage follicles, (ii) forces the follicular transition from stage III to stage IV producing enlarged stage IV follicles, and (iii) induces atresia by producing atretic follicles that are peculiarly enlarged (i.e., big atretic follicles). We suggest that BPA induces atresia by the primary effect on recruitment of stage I follicles. This forces follicular progression and produces stage IV follicles that are peculiarly enlarged that undertake the atretic development.

Primary prevention of cardiovascular disease in patients with systemic lupus erythematosus: case series and literature review.

Background Systemic lupus erythematosus is associated with an increased risk of cardiovascular disease. Low-dose aspirin, hydroxychloroquine and statins have been suggested to play a prophylactic role of cardiovascular events. This study is devoted to reviewing the literature on the topic and assessing the effects of these drugs in preventing a first cardiovascular event in a two-centre Italian series. Methods A PubMed search on cardiovascular prevention in systemic lupus erythematosus was performed. Moreover, systemic lupus erythematosus patients admitted to two centres from 2000-2015, who at admission had not experienced any cardiovascular event, were investigated. Aspirin, hydroxychloroquine and statin use, and the occurrence of any cardiovascular event, were recorded at each visit. Kaplan-Meier and Cox regression analyses were performed to evaluate the role of traditional, disease-related cardiovascular risk factors and of each of the three drugs in the occurrence of new cardiovascular events. Results The literature search produced conflicting results. Two hundred and ninety-one systemic lupus erythematosus patients were included in the study and followed for a median of eight years. During follow-up, 16 cardiovascular events occurred. At multivariate analysis, taking aspirin (hazard ratio: 0.24) and hydroxychloroquine for more than five years (hazard ratio: 0.27) reduced, while antiphospholipid antibody positivity (hazard ratio: 4.32) increased, the risk of a first cardiovascular event. No effect of statins emerged. Conclusion Our study confirms an additive role of aspirin and hydroxychloroquine in the primary prophylaxis of cardiovascular events in Italian patients with systemic lupus erythematosus. The lack of any detected effect in previous reports may depend on the design of studies and their short follow-up period.

Autoimmune gastrointestinal complications in patients with systemic lupus erythematosus: case series and literature review.

The association of systemic lupus erythematosus (SLE) with gastrointestinal autoimmune diseases is rare, but has been described in the literature, mostly as case reports. However, some of these diseases may be very severe, thus a correct and early diagnosis with appropriate management are fundamental. We have analysed our data from the SLE patient cohort at University College Hospital London, established in 1978, identifying those patients with an associated autoimmune gastrointestinal disease. We have also undertaken a review of the literature describing the major autoimmune gastrointestinal pathologies which may be coincident with SLE, focusing on the incidence, clinical and laboratory (particularly antibody) findings, common aetiopathogenesis and complications.

Expression and localization of the deubiquitinating enzyme mUBPy in wobbler mouse testis during spermiogenesis.

Mouse ubiquitin-specific processing protease (mUBPy) is a deubiquitinating enzyme highly expressed in both brain and testis. In testis, it interacts with the DnaJ protein, MSJ-1; both mUBPy and MSJ-1 are located on the cytoplasmic surface of the developing acrosome and in the centrosomal region during spemiogenesis. Present data show the first appearance in testis of mUbpy mRNA and protein at 10 days post-partum (d.p.p.). In addition, to investigate on a possible role of mUBPy in sperm formation, we took advantage of mutant wr/wr (wobbler) mice characterized by male infertility, which is likely due to the lack of a real, functional acrosome. RT-PCR and Northern blot analyses show that mUbpy is up-regulated in adult wobbler testis. Furthermore, in wild-type testis mUBPy protein is primarily detected by Western blot in the soluble (cytosolic/nuclear) fraction during the first round of spermatogenesis and in the adult. By contrast, mUBPy is primarily detected in membranous/insoluble protein fraction when wobbler phenotype is clearly shown (30 d.p.p.) and in adult wobbler testis. By immunohistochemistry, whereas in wild-type animals mUBPy marks the profile of the acrosomic vesicle in differentiating spermatids, in wobbler mice only a detergent pre-treatment procedure allows to detect mUBPy immunoreactivity, which results in diffuse spotted granules inside the cytoplasm and around the nuclear shape. In conclusion, in wobbler testis expression of mUbpy is up-regulated, while a differential sorting of the protein characterizes wobbler spermatids where acrosome formation is impaired.

Endocannabinoid system in first trimester placenta: low FAAH and high CB1 expression characterize spontaneous miscarriage.

Anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were the first endocannabinoids to be characterized, that bind two G protein-coupled receptors, CB1 and CB2. AEA synthesized by multiple pathways, including NAPE-specific phospholipase D (NAPE-PLD) and degraded by the fatty acid amide hydrolase (FAAH). AEA levels are critical in regulating embryo development and the "window" of implantation. We examined the expression of nape-pld mRNA, CB1 and FAAH in human placenta hypothesizing that their altered signaling may contribute to spontaneous miscarriage. First trimester placentas from women with spontaneous miscarriage (group 1) were matched with placentas from women who underwent termination (group 2). Nape-pld expression was analyzed by RT-PCR; CB1 and FAAH expression by Western blot and immunohistochemistry. Nape-pld mRNA expression was higher in group 2 than in group 1. Western blot analysis revealed higher CB1 expression and lower or absent FAAH in group 1 than in group 2. Immunohistochemistry confirmed CB1 and FAAH signals in group 1 and group 2 placentas, respectively. Human placenta contains the enzymes to synthesize AEA. Moreover, placental tissue represents a target for endocannabinoids whose activity may regulate pregnancy outcome. In particular, very low or absent FAAH and high CB1 levels correspond with spontaneous miscarriage.

Multipotential neural precursors transplanted into the metachromatic leukodystrophy brain fail to generate oligodendrocytes but contribute to limit brain dysfunction.

Neural stem cells appear to be best suited for regenerative therapy in neurological diseases. However, the effects of high levels of potentially toxic substances such as sulfatides--which accumulate in metachromatic leukodystrophy (MLD)--on this regenerative ability are still largely unclear. To start addressing this question, in vitro and in vivo experiments were used to examine the behavior of multipotential neural precursors exposed to abnormally high levels of sulfatides. Following transplantation of dissociated neurospheres into the brain of presymptomatic MLD pups, the majority of donor-derived cells were distributed in a caudal to rostral direction, with higher numbers in the cortex. Most if not all of the donor cells acquired an astroglial phenotype. We found no evidence of oligodendrocyte or neuronal commitment of transplanted cells in long-term-treated MLD mice (e.g. up to 1.5 years of age). This was in line with our in vitro findings of sulfatides blocking oligodendrocyte formation after induction of differentiation in sulfatide-treated epidermal growth factor/fibroblast growth factor responsive neurospheres. Transplanted MLD mice showed an improved arylsulfatase A (ARSA) activity and a significant amelioration of sulfatide metabolism, neurodegeneration and motor-learning/memory deficits. Furthermore, transplanted cells were shown to act as a source of ARSA enzyme that accumulated in endogenous brain cells, indicating the occurrence of enzyme cross-correction between transplanted and host cells. These results provide a first insight into the effect of sulfatides on the stemness properties of neural stem cells and on the effects of the MLD environment on the in vivo expectations of using neural stem cells in cell therapy.

Structure of msj-1 gene in mice and humans: a possible role in the regulation of male reproduction.

Msj-1 gene encodes a DnaJ protein highly expressed in spermatids and spermatozoa of both rodents and amphibians, possibly involved in vesicle fusion and protein quality control by means of interaction with heat shock proteins. We isolated and characterized the entire murine msj-1 gene and searched for putative msj-1-like genes into the human genome. Furthermore, ultrastructural localization of MSJ-1 was analyzed in mouse germ cells by immunogold electron microscopy. The analysis of murine msj-1 genomic sequence reveals that it is an intron less gene. Putative promoter region was predicted within the 600 bp upstream the transcription start site. In mouse, msj-1 maps on chromosome 1, into an intronic region of UDP glucuronosyl-transferase 1 family cluster. At ultrastructural level, MSJ-1 marks the developing acrosomic vesicle and the sperm centriolar region. A blast search against the human genome database revealed two closed regions (Ha and Hb) on human chromosome 2 having high nucleotide identity with murine msj-1 coding region. Similarly to mouse, in human both regions map into an intronic region of UDP glycosyl-transferase 1 family polypeptide A cluster (ugt1a@). A significant ORF encoding a putative DnaJ protein of 145 aa was predicted from Ha. Finally, expression analysis, conducted by RT-PCR in human sperm cells, demonstrated that Ha mRNA is effectively present in humans; by Western blot, a specific MSJ-1 band of approximately 30kDa was detected in human sperm. Taken together, these data suggest that msj-1 gene might be conserved among vertebrates and might exert fundamental functions in reproduction.

Safety of arylsulfatase A overexpression for gene therapy of metachromatic leukodystrophy.

Successful gene therapy approaches for metachromatic leukodystrophy (MLD), based either on hematopoietic stem/progenitor cells (HSPCs) or direct central nervous system (CNS) gene transfer, highlighted a requirement for high levels of arylsulfatase A (ARSA) expression to achieve correction of disease manifestations in the mouse model. Full assessment of the safety of ARSA expression above physiological levels thus represents a prerequisite for clinical translation of these approaches. Here, using lentiviral vectors (LVs), we generated two relevant models for the stringent evaluation of the consequences of ARSA overexpression in transduced cells. We first demonstrated that ARSA overexpression in human HSPCs does not affect their clonogenic and multilineage differentiation capacities in clonogenic assays and in a neonatal hematochimeric mouse model. Further, we studied ARSA overexpression in all body tissues by generating transgenic mice overexpressing the ARSA enzyme by LV up to 15-fold above the normal range and carrying multiple copies of LV in their genome. Characterization of these mice demonstrated the safety of ARSA overexpression in two main gene therapy targets, HSPCs and neurons, with maintenance of the complex functions of the hematopoietic and nervous system in the presence of supraphysiological enzyme levels. The activity of other sulfatases dependent on the same common activator, sulfatase-modifying factor-1 (SUMF1), was tested in ARSA-overexpressing HSPCs and in transgenic mice, excluding the occurrence of saturation phenomena. Overall, these data indicate that from the perspective of clinical translation, therapeutic levels of ARSA overexpression can be safely achieved. Further, they demonstrate an experimental platform for the preclinical assessment of the safety of new gene therapy approaches.

Cerebellar neurons and glial cells are transducible by lentiviral vectors without decrease of cerebellar functions.

Due to the profuse connections of the cerebellum to the rest of the central nervous system, cerebellar dysfunction impacts tremendously on movement coordination, maintenance of equilibrium, muscle tone and motor memory. Efficient gene transfer of therapeutic genes to this central nervous system structure would constitute a relevant step ahead the design of treatments to ameliorate cerebellar dysfunction. Lentiviral vectors (LVs) have been used as efficient vehicles to integrate transgenes into dividing and non-dividing cells, such as postmitotic adult neurons, with minimal toxicity and immune response. This study aimed to use LVs carrying green fluorescent protein (GFP) cDNA for transduction of cerebellar cells in vivo without compromising neurological cerebellar functions. Our results indicate that LVs, injected in the lobulus simplex, transduced different cerebellar neurons including stellate, Purkinje cells, granular neurons and glial cells such as astrocytes, oligodendrocytes, and that this gene transfer approach was not accompanied by cerebellar deficits.

Structure of msj-1 gene: a comparative analysis.

Msj-1 gene encodes a DnaJ protein highly expressed in spermatids and spermatozoa of both rodents and amphibians. We isolated and characterized the msj-1 gene in mice. A bioinformatic approach was then used to predict the putative promoter region, chromosomal localization, and its presence in the human genome. The analysis of msj-1 genomic sequence revealed that msj-1 is an intronless gene. Interestingly, two regions (A and B, separated by 10,682 bp) on human chromosome 2 having respectively 78% and 77% nucleotide identity with the murine msj-1 coding region were identified. This suggests the existence of an msj-1-like gene also in humans.

Jun localization in cytosolic and nuclear compartments in brain-pituitary system of the frog, Rana esculenta: an analysis carried out in parallel with GnRH molecular forms during the annual reproductive cycle.

The presence of c-jun like mRNA was assessed in the brain of the frog, Rana esculenta, during the annual sexual cycle. In parallel, Jun protein and GnRH molecular form (mammalian and chicken II also indicated as GnRH1 and GnRH2, respectively) activity was studied in order to establish possible relationships. Northern blot analysis of total RNA reveals the presence of a 2.7 kb c-jun-like mRNA. Western blots, carried out on cytoplasmic and nuclear protein extracts, show the presence of Jun immunoreactive band of 39 kDa in brain and pituitary. Fluctuations of c-jun-like mRNA and Jun immunoreactive protein (cytoplasmic and nuclear) levels in brains during the year indicate relationships among transcription, translation, and nuclear activity. In particular, mRNA levels increase gradually from September until November when Jun protein concentration peaks in cytosolic extracts. Conversely, the nuclear protein reaches highest concentration in July when the cytosolic level shows low values. Immunocytochemical studies confirm the presence of Jun immunoreactivity in both cytoplasmic and nuclear compartments of several brain areas, including those primarily involved in gonadotropin discharge (e.g., anterior preoptic area and preoptic nucleus). GnRH molecular forms and Jun are colocalized in anterior preoptic area and preoptic nucleus. Moreover, during the period characterized by GnRH release, Jun levels strongly decrease in nuclei. Finally, we show that treatments with a GnRH analog (buserelin, Hoechst, Frankfurt) increase Jun levels in brain nuclear extracts.

Cellular mechanisms of striatum-dependent behavioral plasticity and drug addiction.

The striatum has long been known to be involved in the control of motor behavior, since disruption of dopamine-mediated function in this brain structure is directly linked to Parkinson's disease and other disorders of movement. However, it is now accepted that both dorsal and ventral striatal nuclei are also essential for a variety of cognitive processes, which depend on reward-based stimulus-response learning. Since the neuroanatomical and neurochemical organization of dorsal and ventral striatum is only partially overlapping, it is likely that both common and nucleus-specific cellular and molecular events contribute to synaptic plasticity, learning and memory processes mediated by these cerebral structures. Alterations in cell signaling in the striatum may be particularly important in the response to both acute and chronic administration of drugs of abuse, resulting in maladaptive changes in the reward-based associative learning involved in addiction, withdrawal and relapse.

Early defect in the expression of mouse sperm DNAJ 1, a member of the DNAJ/heat shock protein 40 chaperone protein family, in the spinal cord of the wobbler mouse, a murine model of motoneuronal degeneration.

Prevention of protein misfolding is ensured by chaperone proteins, including the heat shock proteins (HSP) of the DNAJ/HSP40 family. Detection of abnormal protein aggregates in various neurodegenerative diseases has led to the proposal that altered chaperone activity contributes to neurodegeneration. Msj-1, a DNAJ/HSP40 protein located around the spermatozoa acrosome, was recently found to be down-regulated in the testis of wobbler mutant mice. Wobbler is an unidentified recessive mutation which triggers progressive motoneuron degeneration with abnormal intracellular protein accumulations, and defective spermatozoa maturation. Here, we examined Msj-1 expression in the spinal cord of the mutants and their controls. Msj-1 transcripts were amplified by reverse transcription-polymerase chain reaction from mutant and wild-type spinal cord RNA. Sequencing of Msj-1 coding region revealed no change in the mutant. In contrast, decreased Msj-1 mRNA levels were observed in five to six-week-old wobbler mice spinal cord, when motoneuron degeneration is at its apex, as compared to controls. A similar decrease was observed in two-week-old wobbler spinal cord, when the number of motoneurons is still unaltered, indicating that the decreased mRNA content is intrinsic to the mutant and not simply related to the loss of cells expressing Msj-1. Assays of Msj-1 protein levels yielded similar results. Immunofluorescent labeling revealed numerous Msj-1-ir motoneurons in five-week-old control spinal cord while no signal was observed in age-matched wobbler. Our results show, therefore, that Msj-1 expression is down-regulated in both organs affected by the wobbler mutation, the CNS and the testis, and that this defect precedes the first histological signs of motoneuron degeneration. These results provide the first example of an association between transcriptional repression of a chaperone protein and a neurodegenerative process.

The amphibian testis as model to study germ cell progression during spermatogenesis.

Testicular morphology of vertebrate testis indicates requirement of local control. In urodeles, the testis is organized in lobes of increasing maturity throughout the cephalocaudal axis. The anuran testis is organized in tubules. Spermatogenesis occurs in cysts composed by Sertoli cells enveloping germ cells at synchronous stages. Moreover, in numerous species germ cell progression lasts a year which defines the sexual cycle. Due to the above quoted features, research on factors regulating germ cell progression in amphibians may reach greater insight as compared with mammalian animal models. In particular, studies on endocrine and paracrine/autocrine factors involved in the regulation of germ cell functions reveal that fos activation and a J protein, previously specifically found in mouse testis, exert an important role in spermatogonial proliferation and maturation of post-meiotic stages, respectively.

Effects of multiple injections of ethane 1,2-dimethane sulphonate (EDS) on the frog, Rana esculenta, testicular activity.

Ethane 1,2-dimethane sulphonate (EDS) is an alkylating agent, which has a selective cytotoxic effect on Leydig cells in some mammalian species. Similarly, in the frog, Rana esculenta, Leydig cells are destroyed after a single EDS injection and regenerate after 28 days. Regeneration of Leydig cells in frogs appears to be independent of the pituitary. The present experiments in R. esculenta were carried out: a) to investigate Leydig cell responsiveness to gonadotropin stimulation during 58 days after a single EDS injection; and b) to assess whether four consecutive EDS injections induce additional effects on the testicular cell population. Our results show that androgen stimulation after gonadotropin injections is restored after 44 days from a single EDS injection. Since the interstitial compartment appears to be normal at least 28 days after EDS treatment, it is likely that new Leydig cells lack gonadotropin receptors. With respect to multiple-EDS injections, Leydig cells completely disappear in several areas and the adjacent germinal compartment is disorganised. In some cases damaged germinal compartment is still surrounded by intact Leydig cells. Surprisingly, testicular and plasma androgens strongly increase in EDS-treated animals. Therefore, Sertoli cells may produce substances inhibiting androgen production in Leydig cells. J. Exp. Zool. 287:384-393, 2000.

Fos localization in cytosolic and nuclear compartments in neurones of the frog, Rana esculenta, brain: an analysis carried out in parallel with GnRH molecular forms.

C-fos activity was determined in the brain of the frog, Rana esculenta, during the annual sexual cycle. The localization of GnRH molecular forms (mammalian- and chicken-GnRHII) was also carried out to determine whether or not the proto-oncogene and the peptides showed a functional relationship. Northern blot analysis of total RNA revealed the presence of a single strong signal of c-fos like mRNA of 1.9 Kb during February and April. This was followed by expression of c-Fos protein (Fos) in several brain areas during March and July shown by immunocytochemistry. In particular, the olfactory region, the lateral and medial pallium, the nucleus lateralis septi, the ventral striatum, the caudal region of the anterior preoptic area, the suprachiasmatic nucleus, the ventral thalamus, tori semicircularis and ependymal layers of the tectum were immunostained. There was no overlap between Fos immunoreactive perikarya and GnRH immunoreactive perikarya (e.g. gonadotrophin-releasing hormone (GnRH) in the rostral part and Fos in the caudal region of the anterior preoptic area). Interestingly, a cytoplasmic localization of Fos was also observed by immunocytochemistry and gel retardation experiments supported this observation. Cytoplasmic extracts from September-October animals bound the AP1 oligonucleotide. The complex was not available in the nuclear extracts from the same preparation, suggesting that, besides Fos, Jun products were also present. Conversely, nuclear but not cytosolic binding was detected in the brain of animals collected in July. In conclusion, we show that Fos and GnRH activity does not correlate in the frog brain and, for the first time in a vertebrate species, we give evidence of a cytoplasmic AP1 complex in neuronal cells.

c-fos activity in Rana esculenta testis: seasonal and estradiol-induced changes.

Estradiol-17beta (E2) is suspected to exert a role in the regulation of testicular activity. Using a nonmammalian vertebrate model (the frog, Rana esculenta), we have investigated whether c-fos activity is detectable in the testis during the annual sexual cycle and whether E2 exerts a regulatory role on spermatogenesis through fos activity. FOS protein is available in testicular nuclear extracts (about 60 kDa) and, surprisingly, also in cytosolic extracts (about 60, 80, and 100 kDa). Estradiol induces primary spermatogonia (ISPG) proliferation [this effect is counteracted by antiestrogens (Tamoxifen and ICI 182-780)] and FOS appearance in testicular cytosolic extracts as well as c-fos transcription. Also, this effect is counteracted by ICI 182-780. Interestingly, the number of FOS immunopositive nuclei of ISPG strongly increases after E2 treatment, whereas a great increase of immunopositivity in the cytoplasm of ISPG is observed with the contemporaneous treatment with antiestrogens. In conclusion, our results demonstrate that E2 induces ISPG multiplication in the frog, R. esculenta, and, for the first time in a vertebrate species, that it triggers c-fos activity in the testis. Moreover, E2 may be involved in mechanisms related to FOS transport in the nucleus of ISPG to induce the mitotic activity.