Alternative activation of hedgehog pathway induced by ultraviolet B radiation: preliminary study.

BACKGROUND: There is still much ambiguity in studies of Sonic hedgehog (Shh) pathways and its dysregulation. Some studies concerning the role of the Shh pathway in basal cell carcinoma (BCC) have been conducted, but there is a lack of studies about Shh pathway dysregulation under the influence of ultraviolet (UV)B radiation. AIM: To evaluate skin expression of Shh, Ptch1, Ptch2, Smo and Gli1 proteins in BCCs with and without the influence of UVB radiation. METHODS: In total, 34 healthy controls (HCs) and 42 patients with nodular BCC were recruited into the study. Patients were divided into five groups (A-E), depending on UVB dose received and BCC status. In all skin specimens, expression of Shh, Ptch1, Ptch2, Smo and Gli1 protein was evaluated. RESULTS: Comparing the BCC group with the HC group, there was significantly higher expression of Shh, Ptch1, Ptch2, Smo and Gli1 proteins. Expression of Ptch2, Smo and Gli1 was increased in response to UVB doses of 3 MED (minimal erythema dose), whereas expression of Ptch1 and Shh was unaffected. CONCLUSION: The lack of change in expression of Shh and Ptch1 after exposure to UVB suggests that the Shh pathway may be activated through a noncanonical pathway under the influence of strong UVB doses.

Expression of the JAK/STAT Signaling Pathway in Bullous Pemphigoid and Dermatitis Herpetiformis.

A family of eleven proteins comprises the Janus kinases (JAK) and signal transducers and activators of transcription (STAT) signaling pathway, which enables transduction of signal from cytokine receptor to the nucleus and activation of transcription of target genes. Irregular functioning of the cascade may contribute to pathogenesis of autoimmune diseases; however, there are no reports concerning autoimmune bullous diseases yet to be published. The aim of this study was to evaluate the expression of proteins constituting the JAK/STAT signaling pathway in skin lesions and perilesional area in dermatitis herpetiformis (DH) and bullous pemphigoid (BP), as well as in the control group. Skin biopsies were collected from 21 DH patients, from 20 BP patients, and from 10 healthy volunteers. The localization and expression of selected STAT and JAK proteins were examined by immunohistochemistry and immunoblotting. We found significantly higher expression of JAK/STAT proteins in skin lesions in patients with BP and DH, in comparison to perilesional skin and the control group, which may be related to proinflammatory cytokine network and induction of inflammatory infiltrate in tissues. Our findings suggest that differences in the JAK and STAT expression may be related to distinct cytokines activating them and mediating neutrophilic and/or eosinophilic infiltrate.

Serum neuron specific enolase - a novel indicator for neuropsychiatric systemic lupus erythematosus?

OBJECTIVE: Neuropsychiatric (NP) lupus, a common manifestation of systemic lupus erythematosus (SLE), is still insufficiently understood, in part, because of the lack of specific biomarkers. Neuron specific enolase (NSE), an important neuronal glycolytic enzyme, shows increased serum levels following acute brain injury, and decreased serum levels in several chronic disorders of the nervous system, including multi infarct dementia, multiple sclerosis and depression. The aim of the study was to evaluate serum NSE levels in SLE patients with and without nervous system involvement, and in healthy controls, and to assess the correlation of NSE serum levels of patients with neuropsychiatric systemic lupus erythematosus (NPSLE) with clinical parameters. METHODS: The study comprised 47 SLE patients and 28 controls. SLE activity was assessed using the Systemic Lupus Activity Measure (SLAM). A neurologist and a psychiatrist examined all patients. NP involvement was diagnosed according to strict NPSLE criteria proposed by Ainiala and coworkers, as modification to American College of Rheumatology (ACR) nomenclature and case definitions. NSE serum levels were determined by use of an immunoassay. RESULTS: Mean NSE serum concentrations in patients with NPSLE were significantly lower than in non-NPSLE patients (6.3 ± 2.6 µg/L vs. 9.7 ± 3.3 µg/L, p < 0.01) and in controls (8.8 ± 3.3 µg/L, p < 0.05). There were significant negative correlations between NSE serum levels and SLE activity (r = -0.42, p < 0.05) and the number of NPSLE manifestations diagnosed (-0.37; p = 0.001). CONCLUSION: Decreased serum concentrations of NSE may reflect chronic neuronal damage with declined metabolism of the nervous tissue in patients with NPSLE.

A multicenter photoprovocation study to identify potential biomarkers by global peptide profiling in cutaneous lupus erythematosus.

Cutaneous lupus erythematosus (CLE) is an inflammatory autoimmune skin disease in which abnormal photosensitivity is an important pathogenetic factor but is difficult to predict, creating a challenge in determining treatment efficacy. Although photosensitivity in CLE patients may change over time, photoprovocation testing with ultraviolet (UV) A and UVB irradiation can be a helpful tool to explore differences between responders and nonresponders during photoprovocation. To identify biomarkers that could substitute for the clinical endpoint lesion development, we performed a global peptidomics profiling analysis of CLE subjects in a controlled photoprovocation study. Plasma and skin biopsy samples were collected before and after UV-irradiation from 13 healthy volunteers and 47 CLE subjects. Twenty-two of the 47 CLE subjects developed skin lesions. The samples were analyzed using a label-free quantitative peptidomics workflow combined with univariate and multivariate statistical analyses. The primary finding was identification of a specific plasma peptide signature separating responders versus nonresponders at baseline. The peptide signature consisted of beta 2-microglobulin (B2MG), human beta-defensin-1, and peptides derived from CD99, polymeric immunoglobulin receptor, and immunoglobulin kappa light chains. In skin, elevated B2MG levels correlated with lesion formation. Our results show that the peptidome is a rich source of potential biomarkers for predicting photosensitivity in CLE.

Exacerbations of bipolar disorder triggered by chloroquine in systemic lupus erythematosus--a case report.

Despite precise definitions and exclusions for 19 syndromes of neuropsychiatric systemic lupus erythematosus (NPSLE), under some circumstances it appears to be difficult to differentiate whether neuropsychiatric symptoms are caused by SLE or by other reasons such as primary mental disorders or substance-induced mood disorders, especially induced by glucocorticoids or antimalarials. We report the case of a male patient with SLE who presented with an exacerbation of bipolar disorder triggered by chloroquine. Firstly, when the patient was diagnosed with SLE, he underwent six months of therapy with chloroquine without any psychiatric symptoms. Later, the SLE returned and the patient was prescribed chloroquine again, without any mental illness. When the third exacerbation of SLE occurred, it coincided with a severe depressive episode with psychotic features that became aggravated for the first time after the administration of chloroquine. The chloroquine was subsequently replaced with hydroxychloroquine for the next six months without any behavioral problems, following which, the SLE and mood disorder were in remission. Later, a bipolar disorder relapse occurred, manifested by a manic episode, and in the following three months, despite psychiatric treatment, a manic episode with psychotic features developed four days after chloroquine was prescribed for arthritis. It was the second time that the mood disorder was exacerbated by chloroquine. Since that time, chloroquine has been withdrawn. Currently the patient is undergoing treatment with hydroxychloroquine and psychiatric drugs with good response. Our case points out that although chloroquine-induced psychosis is rare, patients presenting with behavioral changes need physicians' attention in order to diagnose early and efficiently treat encountered mood disorders.

Toll-like receptor 4 gene polymorphism 1196 C/T does not influence the risk of neuropsychiatric systemic lupus erythematosus in Polish population--a preliminary report.

OBJECTIVE: Recent data indicate that Toll-like receptors (TLRs) participate in various neuropathologic conditions, including ictogenesis, myelin disruptions associated with chronic alcohol abuse, behavioral and cognitive dysfunctions associated with alcohol-induced neuroinflammatory damage, and activation of microglia to reduce amyloid ß deposits. As seizures and depression are well known neuropsychiatric syndromes in systemic lupus erythematosus (SLE) the aim of the study was to investigate whether TLR4 gene polymorphism 1196C/T (rs4986791, Thr399Ile) was a candidate for susceptibility of development of neuropsychiatric systemic lupus erythematosus (NPSLE). METHODS: The study covered 60 patients with SLE and 100 healthy individuals. TLR4 1196C/T genotyping was performed by real-time polymerase chain reaction with the SimpleProbe. RESULTS: The SLE group comprised 86.7% of patients with wild-type homozygotes CC and 13.3% heterozygotes CT and no homozygotes TT. The control group consisted of 85% wild-type homozygotes CC, 15% heterozygotes CT and no homozygotes TT. The frequencies of genotype and allele distribution in SLE patients did not differ significantly from those of the control subjects. The probability of describing the possible risk of SLE imputed to genotype did not significantly differ in comparison with the healthy individuals (p = 0.77, odds ratio = 0.87, 95% confidence interval 0.34-2.19). A significant genotype association of genotype CC with arthritis was found in SLE patients (p = 0.02). It was further confirmed by a significant association of a dominant allele C with arthritis (p = 0.02). No association between CC and CT genotypes of TLR4 1196C/T and NPSLE was found. Allele distribution of TLR4 1196C/T also was not associated with NPSLE. No other significant differences were found in genotype and allele frequencies regarding clinical manifestation of SLE patients. CONCLUSION: In the Polish population of SLE patients, 1196C/T polymorphism of TLR4 gene does not increase the risk of development of NPSLE; however, genotype CC and a dominant allele C is associated with arthritis in the course of SLE.

Vitamin D status in systemic lupus erythematosus patients and its association with selected clinical and laboratory parameters.

OBJECTIVES: To identify relationships between vitamin D serum levels and the presence of autoantibodies directed against vitamin D and levels of interleukin(IL)-17 and IL-23 in patients with systemic lupus erythematosus (SLE). METHODS: The study included 49 patients with SLE. Serum concentrations of 25(OH)D(3) were measured with electrochemiluminescence immunoassay (ECLIA). Enzyme-linked immunosorbent assays (ELISA) were used to determine antibodies directed against 1,25(OH)(2)D(3) and levels of IL-17 and IL-23 in serum of SLE patients. In evaluation of vitamin D status, the control group consisted of 49 age and gender matched healthy individuals, whereas in assessment of anti-vitamin D antibodies the control group comprised 30 sera from blood donors. RESULTS: Serum concentration of 25(OH)D(3) in SLE patients during the warm season was 18.47 ± 9.14 ng/ml, which was significantly decreased as compared with that of the control group - 31.27 ± 12.65 ng/ml (p = 0.0005). During the cold season a trend toward lower concentration of 25(OH)D(3) in SLE patients was revealed; however, it did not reach statistical significance (11.71 ± 7.21 ng/ml vs. 16.01 ± 8.46 ng/ml; p = 0.054). Results within the recommended range for vitamin D (30-80 ng/ml; 70-200 nmol/l) were observed only in three patients. The 25(OH)D(3) concentration was decreased in SLE patients with renal disease or leucopenia as compared with the levels in patients who did not have either problem (p = 0.006 and p = 0.047, respectively). The cold season was found to be a risk factor for vitamin D deficiency (<20 ng/ml) (odds ratio = 9.25; p = 0.005). Autoantibodies directed against 1,25(OH)(2)D(3) were detected in three SLE patients. No significant difference in 25(OH)D(3) serum concentrations was found between SLE patients with and without these autoantibodies. No link was shown between the existence of autoantibodies against 1,25(OH)(2)D(3) and clinical or laboratory findings, including IL-17 and IL-23 levels. However, serum concentrations of IL-23 were lower in patients with vitamin D deficiency (p = 0.037). CONCLUSIONS: SLE patients, especially those with leucopenia or renal involvement, are at high risk of vitamin D deficiency and require vitamin D supplementation. Some SLE patient sera contained 1,25(OH)(2)D(3) antibodies, but these antibodies do not appear to affect vitamin D levels.

Effect of chloroquine phosphate treatment on serum MMP-9 and TIMP-1 levels in patients with systemic lupus erythematosus.

Antimalarials are widely used for the treatment of systemic lupus erythematosus. However, their mechanisms of action have not been fully elucidated. Literature data indicate that matrix metalloproteinases may play a role in the immune response and tissue damage that occur in autoimmune skin diseases. The aim of this study was to determine the effect of 3 months of chloroquine treatment on serum levels of MMP-9 and TIMP-1 in patients with systemic lupus erythematosus. The study group consisted of 25 patients with systemic lupus erythematosus and 25 sex- and age-matched healthy volunteers. Before drug administration, serum levels of MMP-9 and TIMP-1 were determined by enzyme-linked immunosorbent assay. The same procedure was performed after chloroquine treatment. We found significantly higher median serum levels of MMP-9 in patients with systemic lupus erythematosus before therapy (57.20 ng/ml) when compared with controls (44.50 ng/ml) (p < 0.001). After chloroquine therapy the median MMP-9 serum level of systemic lupus erythematosus patients decreased significantly (43 ng/ml; p < 0.001). Before treatment the median TIMP-1 serum level in the patients with systemic lupus erythematosus was significantly higher than in the control group (500 vs. 200 ng/ml; p < 0.001), and after therapy it increased significantly (750 ng/ml TIMP-1; p < 0.001). The results suggest that chloroquine treatment may affect the matrix metalloproteinase network, and this effect may contribute to the immunoregulatory and anti-inflammatory properties of antimalarials.

Systematic administration of chloroquine in discoid lupus erythematosus reduces skin lesions via inhibition of angiogenesis.

BACKGROUND: Discoid lupus erythematosus (DLE) is a chronic cutaneous form of lupus erythematosus, characterized by inflammation and scarring skin lesions, with lymphocyte infiltration and vasodilation. Antimalarial drugs have beneficial therapeutic effects in DLE, partially resulting from their immunomodulating and photoprotective properties. The possible influence of these drugs on angiogenesis has not been previously evaluated. AIMS: To investigate the impact of chloroquine (CQ) treatment on the expression of vascular endothelial growth factor (VEGF, a major regulator of angiogenesis) and CD34 (a transmembrane glycoprotein expressed on endothelial cells and involved in tethering lymphocytes) in patients with DLE. METHODS: A 3-mm skin biopsy was taken from typical skin lesions in 10 people with DLE. Another biopsy was taken from the same area after 3 months of treatment with CQ (250 mg/day). Skin sections were stained with monoclonal antibodies directed against VEGF and CD34. The intensity of epidermal VEGF expression, and the number and area of CD34-positive dermal blood vessels were assessed. RESULTS: CQ treatment induced a reduction in epidermal VEGF expression. It also resulted in a significant decrease in the median number of CD34+ dermal blood vessels (from 219 to 125 vessels per mm(2)). Furthermore the median vessel area was significantly lowered from 9.76 x 10(6) to 6.92 x 10(6) mm(2) per mm(2) of the dermis. CONCLUSIONS: These results indicate that one beneficial effect of CQ treatment in DLE may be due to its antiangiogenic properties.

The influence of antimalarial treatment on IL-1beta, IL-6 and TNF-alpha mRNA expression on UVB-irradiated skin in systemic lupus erythematosus.

BACKGROUND: There are very few data addressing the mechanisms of antimalarial treatment benefit locally within the skin of patients with lupus erythematosus, at the level of cytokine messenger RNA (mRNA) expression. OBJECTIVES: The aim of this study was to evaluate whether 3 months of monotherapy with chloroquine influences the mRNA skin expression of interleukin (IL)-1beta, IL-6 and tumour necrosis factor-alpha (TNF-alpha) in nonirradiated and locally ultraviolet B (UVB) irradiated nondiseased skin of patients with systemic lupus erythematosus (SLE). PATIENTS/METHODS: Skin biopsies were collected from 14 patients with SLE 24 h after irradiation at one site and from an adjacent unirradiated site, before and after 3 months of chloroquine treatment. Messenger RNA levels for IL-1beta, IL-6 and TNF-alpha were determined by relative quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: There were no significant differences in the levels of mRNA cytokine expressions in the unirradiated sites before and after 3 months of chloroquine administration. In the irradiated sites, the expression of all three cytokine mRNA levels was significantly higher than in the unirradiated group, approximately 24 h after irradiation, before chloroquine treatment. Significantly lower expression of IL-1beta, IL-6 and TNF-alpha mRNAs was noted in irradiated skin samples after 3 months of chloroquine treatment. CONCLUSIONS: These results demonstrate the local inhibitory effects of chloroquine on UVB-induced upregulation in the mRNA expression of proinflammatory cytokines in irradiated skin of SLE patients, and provide further insight into the apparent immunomodulatory, anti-inflammatory and photoprotective properties of chloroquine.

Chloroquine treatment reduces the number of cutaneous HLA-DR+ and CD1a+ cells in patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that can be exacerbated by exposure to ultraviolet radiation (UVR). The number and phenotype of antigen presenting cells in the skin play a role in cutaneous immune response generation. Although antimalarials are widely used in SLE treatment, their mode of action is not completely elucidated. The aim of our study was to determine the effect of chloroquine treatment on HLA-DR+ and CD1a+ cell number in locally irradiated (three minimal erythema doses of UVB) and normal appearing skin in SLE patients and healthy subjects. A significantly higher number of HLA-DR+ and CD1a+ cells were found in both locations in SLE patients compared with controls. Following three months of daily chloroquine treatment (250 mg), the HLA-DR+ and CD1a+ cell counts were significantly reduced in both irradiated and unirradiated sites of SLE patients, although still higher than in controls. Chloroquine treatment reduces the number of antigen presenting cells in the skin of SLE patients, and this effect may explain the antimalarials beneficial immunoregulatory and anti-inflammatory properties.

Repeated low-dose ultraviolet (UV) B exposures of humans induce limited photoprotection against the immune effects of erythemal UVB radiation.

BACKGROUND: Exposure of human subjects to ultraviolet (UV) B radiation causes immunosuppression. Most experiments to date have not tested the effects of low daily doses of UVB radiation. OBJECTIVES: To ascertain whether photoprotection against several UV-induced immune effects might develop following repeated exposure. METHODS: Groups of approximately 30 healthy individuals were given whole-body UVB irradiation on each of 10 consecutive days with 0.7 minimal erythema dose, or whole-body irradiation as before followed by a single erythemal UVB dose on a small body area, or irradiated only with a single erythemal UVB dose on a small body area, or were not irradiated. They were sensitized with diphenylcyclopropenone (DPCP) 24 h after the final dose, and skin biopsies collected to assess cytokine mRNA expression and the number of cells with thymine dimers and expression cyclooxygenase (COX)-1 and COX-2. RESULTS: The contact hypersensitivity (CHS) response to DPCP was significantly lower in the three irradiated groups compared with the unirradiated controls, while cutaneous interleukin (IL)-1beta, IL-6, IL-10 and tumour necrosis factor-alpha mRNAs, COX-1 and COX-2 and thymine dimers were all significantly higher. When the single erythemal UVB dose was given following the repeated low exposures, a slight downregulation in cytokine expression and thymine dimer formation was indicated. CONCLUSIONS: The repeated low doses of UVB protected to a limited extent against the effects of an erythemal UVB dose on cytokine expression and thymine dimer formation, but not on CHS or COX enzymes.

In search for new antipsoriatic agents: NAD topical composition.

The aim of the study was to examine the effectiveness of the oxidized form of nicotinamide adenine dinucleotide (NAD(+)), adenosine precursor, in 37 patients suffering from psoriasis. As NAD(+) is known to be relatively unstable, the second goal was to establish the proper conditions for the satisfactory stability of topical NAD(+) composition. In each patient, two matching plaques were selected for the study. Topical treatment with 1 or 0.3% NAD(+) in Vaseline ointment administered twice daily was compared with overnight therapy with 0.1% anthralin applied for 12 h and placebo. The enzymatic method was applied to determine the stability of NAD(+) in Vaseline ointment. After a 4-week application, the reduction in erythema, infiltration and desquamation caused by 1 or 0.3% topical NAD(+) composition was similar to the reduction caused by 0.1% anthralin. It was demonstrated that NAD(+) underwent a considerable decomposition at room temperature, while it was sufficiently stable at 5 degrees C; thus, for a longer use the agent should be stored at fridge temperature. NAD(+) therapy combines good efficacy, cosmetic acceptability and convenient twice-daily application.

The cardiac safety of chloroquine phosphate treatment in patients with systemic lupus erythematosus: the influence on arrhythmia, heart rate variability and repolarization parameters.

Antimalarials are used to treat cutaneous and systemic lupus erythematosus (SLE). Even though cardiac damage is a rare complication, over the last decade several reports have raised the issue of cardiotoxicity associated with antimalarials. Therefore, the aim of study was to evaluate the influence of seven-month chloroquine treatment with a 250mg daily dose on arrhythmia, conduction disturbances as well as heart rate variability and repolarization parameters assessed in 24-hour Holter monitoring. The studied group included 28 SLE patients treated with chloroquine as a monotherapy. In all the patients standard 12 leads surface ECG (50 mm) and the 24-hour ECG Holter monitoring (Oxford Medilog Excel-2) were performed before and after chloroquine phosphate treatment. All subjects presented sinus rhythm both at the enrollment and after treatment. No episodes of paroxysmal arrhythmias or conduction disturbances were reported during the study. All the patients were characterized by tendency to tachycardia, but no significant differences in mean heart rate were found before and after chloroquine administration. Similarly, no changes in heart rate variability or repolarization parameters were observed.

Chloroquine treatment influences proinflammatory cytokine levels in systemic lupus erythematosus patients.

Systemic lupus erythematosus (SLE) is an autoimmune disease with a complex pathogenesis. Published data have revealed that serum levels of proinflammatory cytokines are increased in SLE patients. The aim of our study was to evaluate whether monotherapy with chloroquine phosphate affects IL-1beta, IL-6, IL-18 and TNF-alpha serum levels in SLE patients. The study group consisted of 25 SLE patients with mild or moderate disease activity and 25 age- and sex-matched healthy control subjects. In SLE patients the cytokine levels were measured just before and three months after starting chloroquine treatment at a dose of 125 mg twice daily. Although the majority of SLE patients had a low systemic lupus activity measure (SLAM) index, the levels of IL-6, IL-18 and TNF-alpha were significantly higher than in the control group. After three-months of chloroquine therapy the mean level of IL-6, IL-18 and TNF-alpha decreased significantly. Minimal erythema doses (MEDs) were significantly increased in SLE patients after three months of chloroquine therapy. The results indicate that chloroquine treatment lowers some proinflammatory cytokines and may provide a photoprotective effect.