Arch Dermatol Res DOI 10.1007/s00403-011-1133-0
ORIGINAL PAPER
Antigens from Leishmania amastigotes inducing clinical remission of psoriatic arthritis J. A. O’Daly • J. Gleason • R. Lezama • P. J. Rodriguez • E. Silva • N. R. Indriago
Received: 3 December 2010 / Revised: 25 January 2011 / Accepted: 27 January 2011 ! Springer-Verlag 2011
Abstract A first generation vaccine (AS100-1) was manufactured with protein from four cultured Leishmania species, which proved to be effective in the treatment of psoriasis. A single blind trial on 3,132 psoriasis patients revealed 508 (16.2%) subjects with psoriatic arthritis (PsA) that received AS100-1 antigens. The study group was distributed according to percent psoriasis area and severity index (PASI) reduction from PASI 10 to PASI 100. All groups decreased in arthritis score (AS), tender joints counts and nail changes after treatment; the highest decreased in the PASI 100 group. Relapses of psoriasis and PsA had PASI and AS lower than initial values before treatment. Clinical remissions were at lower doses and less time, after the second course of treatment. Peripheral blood mononuclear cells (PBMC) lymphocyte subsets (LS) varied with PASI range (1–10, 11–20 and 21–72). Pre-treatment, absolute values of gated LS: CD4?, CD8?HLA-, CD8?HLA?, CD8?CD3-, CD8?CD3? decreased in PBMC as PASI increased, suggesting migration from the blood to the skin. In contrary to the previous finding, the following LS: CD8?CD4-, CD3?CD8-, HLA?CD8-, CD19, CD8?CD4? and membrane surface immunoglobulin IgA?, IgD?, IgM?, IgE?, and IgG? increased in J. A. O’Daly (&) ! J. Gleason Astralis Ltd., 1076 Stuyvesant Ave., Irvington, NJ 07111, USA e-mail:
[email protected] J. A. O’Daly ! R. Lezama ! P. J. Rodriguez ! E. Silva ! N. R. Indriago Center for Psoriasis Research and Treatment, Ave. Las Ciencias, Calle Codazzi, Los Chaguaramos, Caracas, Venezuela J. A. O’Daly Instituto Venezolano de Investigaciones Cientı´ficas Altos de Pipe, Km. 10 Carretera Panamericana, Caracas 1010A, Venezuela
PBMC as PASI increased suggesting activation and proliferation by unknown antigens creating a homeostatic cycle between skin/joints and peripheral blood. After nine doses of AS100-1, the following LS: CD8?CD3?, CD8?HLA?, CD3?CD8-, CD4?CD8-, CD8?HLA-, HLA?CD8-, CD8?CD3-, CD19?, CD8?CD4-, CD8?CD4?, IgA?, IgD?, IgM?, IgE?, and IgG? decreased significantly as compared with values before treatment. The LS decreased stops the vicious cycle between skin/joints and blood explaining clinical remission of lesions. Keywords Psoriatic arthritis ! Psoriasis ! Immunotherapy ! Leishmaniasis ! Amastigote antigens ! Clinical trial
Introduction While treating subjects in Venezuela with a vaccine (AS100-1) for cutaneous Leishmaniasis (CL), we observed 100% clinical remission of a psoriatic lesion in one subject. A first generation polyvalent vaccine (AS100-1) was manufactured with protein from four cultured Leishmania species. A double-blind, placebo-controlled, parallel group study of multiple doses of AS100-1 was performed on psoriatic subjects, to confirm safety and efficacy [41]. Treatment of plaque psoriasis was conducted in 2,770 subjects and included plaque (79%), guttate (10%), plaque and guttate (10%), palm/plantar (0.3%), erythrodermia (1.8%), inverse (0.8%), plaque and arthritis (3.4%) and nail psoriasis (0.3%). Baseline PASI compared with posttreatment values were: PASI 100, 23%; PASI 75, 45%; PASI 50, 13%; PASI 10, 9%;\PASI 10, 3% while 7% quit treatment. There were no serious adverse events attributed to the treatment drug. Some patients with PsA benefited
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after treatment [41]. To determine the effective factor, a single blind trial with four monovalent second generation vaccines (AS100-2) was performed in 26 subjects, which also had remission of psoriasis. AS100-2 vaccines were further purified, resulting in seven chromatography fractions (AS200) per species. Subsequently, a single-blind trial in 55 subjects treated with a third generation vaccine AS200 prepared with DEAE chromatography fractions from L(L)brasiliensis also induced remission of psoriasis. Interestingly, two HIV? subjects with plaque psoriasis experienced remission after treatment with AS100-1 [40]. AS200 Leishmania antigenic fractions induced linear delayed type hypersensitivity (DTH) reactions over a 1–40 lg dose range in guinea pigs, allowing us to structure a potency assay for the drug product [39]. Psoriasis and PsA are chronic life-long inflammatory diseases. In Europe, the prevalence of psoriasis varies from 0.6 to 6.5%, while in USA is 3.15% [9]. Psoriasis and PsA are less prevalent in China (0.23% for men, 0.16% for women) and Japan than in Europe. Different to Caucasians, the prevalence of psoriasis in Taiwanese people is higher in men than in women increasing significantly in patients over 70 years of age [11]. The estimated prevalence of PsA using the classification criteria for psoriatic arthritis (CASPAR) [8, 10, 16, 44, 55] in the psoriasis population in England was 13.8% [27]. In Finland for PsA, an incidence in adults of 6 and 6.8 per 100,000 was reported in 1990 and 1995, respectively, data similar in southern Sweden. Annual incidence of PsA in Rochester, Minnesota from 1982 to 1991 was 6.59 per 100,000, and in Olmsted County, increased from 3.6 between 1970 and 1979, to 9.8 between 1990 and 2000 [24]. PsA is defined as an immunologically triggered, chronic inflammatory joint disease, associated with cutaneous psoriasis. One-fourth to one-third of all patients develops musculoskeletal involvement typical of PsA [1]. Skin involvement precedes joint symptoms in 80% of cases, although 15% of PsA patients experience joint symptoms first [3]. About 20–40% of patients with psoriasis develop the joint symptoms of PsA, usually seronegative for rheumatoid factor (RF) and for cyclic citrullinated peptide antibody (anti-CCP) [1, 5]. Five types of PsA have been proposed: distal interphalangeal joint only, asymmetrical oligoarthritis, polyarthritis, spondylitis and arthritis mutilans. Classic PsA consists of oligoarthritis, distal interphalangeal joint involvement, dactylitis, and calcaneal enthesitis [19, 26, 36]. About 50% of patients with psoriasis have distinctive nail changes; the most common are pitting, onycholysis, oil spots and dystrophy similar to that observed in onychomycosis, most commonly in patients with PsA [6]. The genes involved in psoriatic arthritis are HLA genes of class I MHC, on the HLA-B and HLA-C loci. Psoriasis
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is linked to HLA-Cw6 allele [1]. Seven loci have been identified associated with psoriasis HLA-C, IL12B, IL23R IL23A, IL4/IL13, TNFAIP3, and TNIP1 [18]. Twenty percent of PsA patients with peripheral joint involvement displayed HLA-B27, a value that climbs to 70% in patients with spine involvement [61–63]. The considerable overlap in associated HLA antigens for both diseases (B13, B17, B57, Cw6, and DR7) suggests a shared genetic predisposition [2, 15]. The inflammation in psoriasis and PsA has been explained by T cell-directed autoimmunity against a common unidentified autoantigen in the skin and the synovial membrane or cartilage. A new model of autoinflammation has been developed linking psoriasis, PsA, and nail involvement. The enthesis forms the synovio-entheseal complex (SEC) around the distal interphalangeal joint [32]. The frequent microdamage and tissue repair at normal SEC attachment sites in healthy joints lead to regional innate immune activation and persistent inflammation, as an alternative to explain PsA. It has been proposed the concept of autoinflammation, whereby tissue-specific factors, including microtrauma, lead to regional innate immune activation and persistent inflammation, different to primary immunopathology driven by T and B cell abnormalities. Distal interphalangeal (DIP) joint disease in PsA is associated with diffuse inflammation that envelops the nail root and adjacent bone [33]. PsA typically presents inflammation at the sites of attachment of tendons, ligaments, and joint capsules to bone (enthesitis). The manifestation at insertions of tendons, ligaments and capsules is typical for psoriatic arthropathy; with the insertion of the Achilles tendon being most commonly involved [36, 37]. PsA is an autoimmune disease in which the CD8? T cell plays a central role, with a predominance of clonally expanded CD8? T cells in the synovial tissue. The dominance of CD8? T cells in PsA synovial fluid suggests that they rather than CD4? lymphocytes are driving the immune response in the joint. Analysis of CD8? ab TCR repertoire in paired synovial fluid and peripheral blood of patients with active PsA found oligoclonal expansions in the TCR-b chain, some of which were shared between simultaneous samples of synovial fluid and peripheral blood [12]. Furthermore, CD8? T cells independent of CD4? T cells may be driving the immunological response, as indicated by the development of PsA in advanced AIDS, different from rheumatoid arthritis (RA) and systemic lupus erythematosus, which are ameliorated by the loss of CD4? T cells. Interestingly, PsA developed after syngeneic bone marrow transplantation from a psoriasis donor [23]. In previous work, we also found that Leishmania amastigote antigens induced clinical remission of psoriasis and PsA in HIV? patients with AIDS which supports the role of CD8? T cells in the PsA inflammatory process [40].
Arch Dermatol Res
Predominant CD8? T cell clones in different joints and at different times are found in PsA implicating the adaptive immune response in the disease [17]. However, these expanded clones lack common structural motifs that would imply the presence of an antigen driven mechanism, which is not consistent with a simple driver clone hypothesis. Furthermore, there is a background of non-clonally expanded polyclonal T cells, reflecting the attraction of the T cells by chemokines to the joints. A striking feature in the tissue and joint fluid is the extreme clonal expansion that persists even during treatment with methotrexate. There is no evidence to indicate that one or a few structurally or cognitively related T-cell clones drive the process, as is the case for most autoimmune diseases. Rather, it appears that a succession of different clones sequentially dominate the repertoire of infiltrating T cells [23]. A third CD4? T cell effector subset, besides Th1 and Th2 CD4? T cells, has been described in peripheral blood mononuclear cell (PBMC) of patients with ankylosing spondylitis (AS) and PsA, named Th17 cells based on their ability to secrete interleukin-17 (IL-17). Through the ubiquitously expressed IL-17 receptor, IL-17 induces the secretion of proinflammatory cytokines, stimulates osteoclast formation and bone resorption, recruits neutrophils and monocytes, and triggers the production of granulocyte– macrophage colony-stimulating factor. Th17 cells are found in the host defense against extracellular bacteria and fungi in mice and, in the antifungal immune response in humans and also Th17 cells are involved in collagen induced arthritis, a classic model of RA [28]. Synovial tissue in PsA is characterized by a sub-lining infiltrate with CD8? T cells and B cells, vascular proliferation and a relatively thin lining layer of proliferating intimal synoviocytes [17]. Angiogenesis, expression of vascular endothelial growth factor (VEGF), and its receptors, dendritic cells (DC), endothelial activation and neutrophil infiltration are prominent features in PsA synovial tissue. Patients with active PsA had significantly higher levels of VEGF compared to patients with inactive PsA and healthy individuals [22]. Synovial tissue in PsA is characterized by the expression of pro-inflammatory cytokines, including interleukin1b (IL1b), interferon-c, tumor necrosis factor alpha (TNFa), IL-6, IL-12, IL-15, IL-17 and IL-18 [23, 46]. Other authors also demonstrated CD8?CD3? T cells (CD8low) with an activated effector phenotype and oligoclonality. The CD8low T cell population may represent a long-term, low-level response driven by chronic antigen exposure representing an immune response to intracellular pathogens [57]. Purified Leishmania antigenic fractions (AS200) induced linear DTH reactions in guinea pigs. A DBA-1 mouse collagen-induced arthritis (CIA) model was used to compare AS200 treatment against: a polyvalent vaccine
(AS100-1), a monovalent vaccine (AS100-2) and placebo. AS100-1 decreased inflammation in mice forepaws and AS200-treated mice had the least amount of forepaw inflammation and the lowest mean arthritis scores, inducing clinical remission of mice arthritis [39]. In this paper, we present evidence of clinical remission of PsA in psoriatic patients after treatment with AS100-1 polyvalent vaccine, together with flow cytometry data that points out the role of CD8? T cells in the PsA inflammatory process.
Materials and methods Materials Aluminum hydroxide low viscosity gel (Rehydragel) was purchased from Reheis Inc., New Jersey. Lowry and bicinchoninic acid (BCA) kits were purchased from SigmaAldrich, USA. The following materials were used for the flow cytometer study: K3 EDTA Vacutainer blood collection tubes, Simultest negative control c1/c2a (IgG1 FITC/IgG2a PE), Simultest LeucoGATE CD45/CD14 (Anti-HLe-1 FITC/Leu-M3PE), Simultest CD4/CD8 (Leu3aFITC clone SK32a/PE clone SK1), and a panel of murine monoclonal antibodies stained with fluorescein isothiocyanate (FITC) or phycoerythrine (PE): CD4 Leu-3aFITC, CD8 Leu2AFITC, CD3 Leu4PE, HLADR PE, CD19 LeuI2FITC, that together with FACS Lysing solution, CaliBRITE beads, and a FACSorter cytometer, were purchased from Becton–Dickinson, San Jose, CA, USA. Preparation of the polyvalent immunogen (AS100-1) The first-generation polyvalent vaccine AS100-1 was prepared with four leishmania species: L.(L)amazonensis, L.(L)venezuelensis, L.(V)brasiliensis and L.(L)chagasi. The final AS100-1 drug substance, contained 1 mg/mL or 250 lg of each Leishmania spp. in phosphate buffered saline (PBS) supplemented with Rehydragel (RH) at 0.25 mL/mg (v/w) of parasitic protein and 4 lg/mL of gentamicin. Each step in the preparation of the immunogen was checked for sterility as previously published [40, 41]. Protein concentration was determined by Lowry or BCA [30, 53]. Statistical methods The analysis was similar to previously published guidelines used for AS100-1 trials [40–42].The primary efficacy endpoint was defined as the percentage reduction from baseline in PASI score after each dose of AS100-1. The ANOVA assumption of normality and homogeneity of
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variances was tested and, where appropriate, a nonparametric approach (Tukey’s test) or Mann–Whitney test was used to compare treatment groups. A last-observationcarried-forward (LOCF) algorithm was used to replace missing observations. All calculations were done with GraphPad Prism software. AS100-1 clinical trial Main criteria for trial inclusion and exclusion were similar to previous AS100-1 trials. Briefly, exclusion criteria consisted of a positive Leishmania infection status and a positive intradermal reaction skin test for Leishmania at screening, evidence of pregnancy or lactation, any immunodeficiency, positive HIV status and opportunistic infections. A limited exception was made to allow treatment of two HIV? subjects. A washout period of 2 weeks for topical treatment and 4 weeks for systemic treatment was applied to all patients as published [40–42]. PASI at baseline was determined at the first visit prior to the subject receiving an intradermal Leishmania test preparation to evaluate the DTH reaction 48 h later. During the second visit the first AS100-1 vaccine, consisting of a 500 lg injection, in 0.5 mL was administered; all subsequent injections were administered bi-weekly at the same dose level as published. Based on the PASI score, changes after each doses of AS100-1 were classified as follows: \10% PASI reduction (PASI between 1 and 9), 10% PASI reduction (PASI between 10 and 49), 50% PASI reduction (PASI between 50 and 74), 75% PASI reduction (PASI between 75 and 89) and 100% PASI reduction (PASI between 90 and 100%) [40–42]. The total number of subjects with psoriasis was 3,132 volunteers. From that group, 508 subjects (16.22%) had PsA that meet the CASPAR criteria [10, 55] and were chosen for the analysis of the present data. The study was conducted at one medical center from March 1992 through May 2002 and included all nine types of psoriasis. The increase in 362 subjects all included in the AS100-1 clinical trial did not change the statistical values already published [41]. The expanded study group had average age of 40.5 ± 15.0 years, 2,894 (92.4%) were between 26 and 65 years of age with a range of 5–88 years. 1,096 subjects (35%) had family histories of psoriasis. The average duration of disease in all subjects was 11.5 ± 10.3 years, ranging from 2 to 62 years, similar in both males and females. The 508 group with PsA had average age of 44.9 ± 14.3 years, range 5–83 years, time with psoriasis 13.2 ± 12.4 years, range 0.4–64 years, 53% females. RF determined in 3% of patients (n = 15) with PsA were negative, a low number, due to economic reasons. 11% (53/508) had diagnosis of psoriasis by skin biopsy, the rest
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by clinical criteria by an experienced dermatologist, checked by a rheumatologist in 15% of cases. Efficacy and safety criteria Efficacy of AS100-1 trial was assessed by performing skin examinations and recording PASI parameters. The primary efficacy parameters were the percentage reduction in PASI score at each visit and the comparative proportions of subjects with 100, 75 and 50% PASI reduction in each treatment group. Safety criteria for the treatment of psoriasis in all trial subjects were evaluated by assessing vital signs, injection site reactions, and physical examinations. In a selected group of few subjects, due to economic reasons, safety was further assessed by clinical laboratory tests as published [40–42]. AS100-1 clinical trial flow cytometry analysis The flow cytometry study followed published guidelines [42]. Pre-treatment blood collection was performed in subjects distributed to one of three groups, according to PASI value: group I (PASI: 1–10), group II (PASI: 11–20) and group III (PASI: 21–72). The post-treatment blood collection was done after each dose (1–9 doses) of AS1001. The three groups consisted of: pretreatment (n = 29), post-treatment (n = 37) and control groups (n = 35). The pre-treatment group had an average age of 47.6 ± 12.5 years, age range between 19 and 78 years, 55.2% were females, time with psoriasis 19.5 ± 12.5 years, range 2–45 years; average initial PASI 15.1 ± 13.6 range 1.2–48 units. The post-treatment group had an average age of 43.1 ± 12.8 years, age range between 18 and 65 years, 59.4% were females, time with psoriasis 12.8 ± 10.1 years, initial PASI 10.8 ± 9.2 units, range 0.4–48 units. The control group had an average age of 38.9 ± 9.1 years, range between 19 and 60 years, 69% females, members of the Venezuelan Institute of Scientific Investigations (IVIC) personnel with no recorded history of Leishmania infection [42]. Immunofluorescence staining and flow cytometry analysis The methods for immunofluorescence staining and quantification of lymphocyte subsets in PBMC have been published [42]. Briefly, all fixed samples were processed in a FACSorter within 8 h after venipuncture. Two-color flow cytometry was performed using a FACSorter cytometer and the data were analyzed using Lysis II. The FACSorter was prepared for sample analysis using CaliBRITE beads and AutoComp software to setup PMT voltages. Subsequently, LeucoGate (CD45/CD14) with SimulSet software was used to create a lymphocyte acquisition gate. The lymphocyte analysis gate parameter(s) included 98% of the
Arch Dermatol Res
normal mature lymphocytes. If the analysis gate contained 3% monocytes the software automatically reduced the light-scatter gate to 95% lymphocytes contained in the sample. A Simultest c1/c2a negative control was run with each sample to set fluorescence-1 (FL1) and fluorescence-2 (FL2) quadrant markers around the unstained (negative) lymphocyte population and to estimate the amount of isotype antibody binding (nonspecific specific staining) caused by Fc receptors, between positively and unstained events in the lymphocyte gate. The phenotypic panel used to stain lymphocyte subsets (LS) in these experiments consisted: MurineIgG1FITC/IgG2aPE, CD45Hleu1FITC/ CD14LeuM3PE, CD8Leu2APE/CD4-Leu3AFITC, CD8Leu2AFITC/CD3-Leu4PE, CD8-Leu2AFITC/HLA-DRPE, CD19LEUI2FITC, anti-mouse poly Ig FITC as negative control, anti-human IgA FITC, antihuman IgD FITC, antihuman IgE FITC, anti-human IgG FITC, anti-human IgG FITC, anti-human IgM FITC. In each stained blood sample, 5,000 events were acquired. Absolute counts for each LS were calculated similar to CD4?LS as example as published [42]: CD4 þ cells lL#1 ¼
% CD4 þ gated lymphocytes 100 % stained lymphocytes % 100 % WBC mm#3 :
Evaluation of psoriatic arthritis Time of onset of skin disease and arthritis, dactylitis, diffuse enthesis pain, inflammatory heel pain, clinical sacroiliitis, family history of psoriasis, inflammatory spinal pain (lumbar, thoracic and cervical), rheumatoid nodules, primary clinical diagnosis, other rheumatic diseases, number of joint surgeries, joint counts (tender, swollen and damaged), and previous treatment were recorded. Radiographic and laboratory analysis as acute-phase reactant levels, RF and antiCCP were limited due to economic reasons. The score for each joint inflammation is presented in Table 1 [52].
Table 1 Arthritis score inflammation value for each joint
Location
Points
Finger
1
Knee
3
Ankle
2
Hip
4
Wrist
2
Elbow
3
Dactylitis
1
Shoulder
3
Vertebral Column Temporo-maxilar jaw
5 2
Definitions used in the protocol were as follows: actively inflamed joints were defined as the number of joints with tenderness and/or swelling. Radiographic damage to peripheral joints was recorded in 2% of cases only, due to economic reasons. All items included in the CASPAR criteria [10, 55], namely presence of inflammatory articular disease, current psoriasis, personal history of psoriasis, family history of psoriasis, psoriatic nail dystrophy, presence of current dactylitis, and history of dactylitis, were recorded at each visit. Inflammatory articular disease (peripheral joints, spine or entheseal) was evaluated with an arthritis score derived from the summation of all values found ad hoc in each patient as described in Table 1 [52].
Results This study was an open label, single center study, which evaluated the safety and efficacy of multiple 500 lg doses of AS100-1 on PsA. The primary objective was to evaluate the safety profile. The secondary objective included the assessment of the optimal doses necessary to induce clinical remission of PsA and psoriasis or zero AS score and PASI. Approximately 2,599 subjects (83%) experienced at least one adverse event (AE). The most frequent AE were injection site related, and included the following: pain 43%, nodule formation 23%, heat 21% and erythema 14%, as previously published [40, 41]. There were no age or gender differences observed for AE, and no deaths occurred during the study. All AE resolved without intervention, usually within 24–72 h. When baseline PASI values in the group with PsA (n = 508) were compared with post-treatment values, clinical remissions were: PASI 100 in 275 (54.1%), PASI 75 in 117 (23%), PASI 50 in 73 (14.4%), and PASI 10 in 43 (8.5%) of subjects, respectively. The average number of AS100-1 doses required to induce 100% remission of psoriasis was 9.9 ± 4.8 (Table 2). One-way ANOVA comparing initial PASI values among the four groups had P B 0.0001 means significantly different (P \ 0.05), R2 = 0.042. Tukey’s comparison tests gave significant differences (\0.05) between PASI 100 versus PASI 75, PASI 100 versus PASI 50 and PASI 100 versus PASI 10. Comparison of initial PASI by twotailed t test between PASI 100 versus 75 (P = 0.001), PASI 100 versus PASI 50 (P = 0.012), PASI 100 versus PASI 10 (P = 0.001) showed a significant difference among groups (Table 2). One-way ANOVA to compare doses for PASI reduction among the four PASI groups showed P = 0.001, mean values were significantly different (\0.05), R2 = 0.031.
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Arch Dermatol Res Table 2 Percent PASI reduction of psoriasis in the group of psoriatic arthritis patients after treatment with AS100-1 (n = 508), initial PASI and doses for PASI reduction in each PASI group % PASI reduction
Subject number, n (%)
Average ± SD Initial PASIa
Doses for PASI reductionb
PASI 10
43 (8.5)
10.7 ± 11.0
7.7 ± 3.9
PASI 50
73 (14.4)
14.2 ± 15.2
10.2 ± 6.1
PASI 75 PASI 100
117 (23.0) 275 (54.1)
13.9 ± 13.2 19.7 ± 17.5
11.2 ± 4.5 9.9 ± 4.8
Statistically significant differences (P \ 0.05) between initial PASI values and doses for PASI reduction a
PASI 100 versus 75, 100 versus 50, 100 versus 10
b
PASI 100 versus 75, 100 versus 10, 75 versus 10, 50 versus 10
Tukey’s multiple comparison test showed significant differences (\0.05) between PASI 100 versus PASI 10, PASI 75 versus PASI 10, PASI 50 versus PASI 10. Patients with the highest initial PASI (19.7) had the best response to Leishmania antigens and needed the lowest number of AS100-1 doses (9.9). Comparison of doses by two tailed t test between PASI 100 versus PASI 75 (P = 0.017), PASI 100 versus PASI 10 (P = 0.004), PASI 75 versus PASI 10 (P B 0.0001), PASI 50 versus PASI 10 (P = 0.019) were all statistically significant (Table 2). The frequency of DIP joint inflammation from hands was three times higher than toes at all PASI values. Inflammatory heel pain, joint deformity and clinical sacroiliitis were more frequent in PASI 100 subjects than in the other PASI groups. Temporo-maxillar jaw arthritis that made the eating process very difficult was present in PASI 100 and 75 groups, respectively. Four patients from the PASI 100 group in wheelchairs could walk again after remission of PsA lesions with AS100-1 treatment (Table 3). Clinical evolution of arthritis score, tender joint counts and nail pathological changes before and after treatment with AS100-1 in the study group (n = 508) revealed a significant decrease in arthritis score from 15.3 to 0.8 after 10.7 doses; in tender joints counts from 7.3 to 0.7 with 10.9 doses and in nail changes from 19 to 4.1 with 14 doses of polyvalent vaccine (Table 4).
One-way ANOVA for AS values before treatment at different PASI values were all similar, no significant differences between PASI groups (Table 5). One-way ANOVA comparing doses to achieve AS lower values after treatment had P = 0.006, mean values significantly different (P \ 0.05) R2 0.02820. Tukey’s multiple comparison tests were significant for PASI 75 versus PASI 50 only. Analysis of doses by two-tailed t test was as follows: between PASI 100 versus PASI 75 (P = 0.045), PASI 100 versus PASI 50 (P = 0.039), PASI 75 versus PASI 50 (P = 0.005), PASI 75 versus PASI 10 (P = 0.018) were all significant (Table 5). Tender joint frequency before treatment gave no significant differences among the PASI groups which means that PsA as measured by joint inflammation counts is evenly distributed among different PASI groups (Table 5). One-way ANOVA comparison of doses to achieve lower tender joint values after treatment had P = 0.002, mean values significantly different (P \ 0.05) R2 0.047. Tukey’s multiple comparison tests were significant for PASI 100 versus PASI 50 and PASI 75 versus PASI 50. The values for PASI 100 versus PASI 50, P = 0.004; PASI 75 versus PASI 50, P = 0.0006; PASI 75 versus PASI 10, P = 0.021 were all significant, which means that higher number of doses are needed to achieve lower tender joint values at the PASI 100 and PASI 75 groups (Table 5).
Table 3 Clinical signs evaluated in the PsA study group (n = 508) distributed according to PASI before treatment with AS100-1 Feature evaluated
Patients (%/508) PASI 10
PASI 50
PASI 75
PASI 100
Total study group independent of PASI
Any hand DIP joint involved
34 (6.3)
58 (11.4)
95 (18.7)
97 (19.1)
284 (55.5)
Any toe DIP joint involved
5 (1)
19 (3.7)
30 (5.9)
28 (5.5)
82 (16.1)
Inflammatory heel pain
0
3 (0.6)
5 (1)
32 (6.3)
40 (7.9)
Any arthrosis or deformity in joints
0
4 (0.8)
4 (0.8)
15 (3.0)
23 (4.6)
Clinical sacroiliitis
0
0
5 (1.0)
15 (3.0)
20 (4)
Temporo-maxilar jaw involved
0
0
1 (0.2)
1 (0.2)
2 (0.4)
Walking in wheel chairs
0
0
0
4 (0.8)
4 (0.8)
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Arch Dermatol Res Table 4 Clinical evolution of arthritis score (AS), tender joint counts and nail pathological changes before and after treatment with AS100-1 in the study group (n = 508) Arthritis scorea Pre-treat
Tender jointsb Post-treat
a
Pre-treat b
Nail changesc Post-treat
Pre-treat c
Post-treat
AS (n, %)
Lower value
Doses
TJC (n, %)
Lower value
Doses
NNC (n, %)
Lower value
Doses
15.3 ± 8.5 (508, 100)
0.8 ± 2.4
10.7 ± 5.1
7.3 ± 3.7 (361,71)
0.7 ± 2.0
10.9 ± 5.0
19.0 ± 8.3 (187, 37)
4.1 ± 5.1
14.0 ± 6.0
Values represent average ± SD All 508 patients had arthritis, 361 had tender joints, and 187 nail changes a
Arthritis score (AS), btender joints counts (TJC), cnumber of nails with changes calculated for each patient as in Table 1 and values averaged in 508 whole group
Table 5 Clinical evolution of arthritis score (AS), tender joint counts and nail pathological changes before and after treatment with AS100-1 in the study group (n = 508) % PASI reduction
10 50 75 100
Arthritis scorea
Tender jointsb
Pre-treat
Post-treat
AS (n, %)
Lower value
13.3 ± 6.1 (43, 8)d
1.7 ± 3.3
15.2 ± 9.3 (73, 14) 16.0 ± 7.6 (117, 23) 15.4 ± 9.1 (275, 54)
Pre-treat
Post-treat
TJC (n, %)
Lower value
9.3 ± 4.5e 7.5 ± 3.2 (37, 7)
Doses
Pre-treat
Post-treat
NNC (n, %)
Lower value
Doses
0.9 ± 1.9
9.5 ± 4.5 20.0 ± 9.3 (8, 2)
5.0 ± 5.8 11.0 ± 3.4f
7.7 ± 3.3 (50, 10)
0.8 ± 1.6
8.7 ± 4.7 20.0 ± 9.8 (22, 4)
3.8 ± 4.7 11.8 ± 6.5f
7.3 ± 3.0 (84, 17)
0.9 ± 2.2 12.2 ± 5.4 19.7 ± 7.9 (35, 7)
7.0 ± 5.2 14..9 ± 5.9f
9.2 ± 6.1
e
0.9 ± 2.4 11.9 ± 5.4
e
0.5 ± 1.9 10.8 ± 4.7
e
7.2 ± 4.2 (190, 37) 0.5 ± 1.4 11.1 ± 4.9 18.6 ± 8.3 (122, 24) 3.3 ± 4.5 13.8 ± 5.4f
1.4 ± 3.8 d
Doses
Nail changesc
Values represent average ± SD Statistical significant values (P \ 0.05) a
Arthritis score (AS) calculated for each patient as in Table 1 and values averaged in each PASI group. Total ? patients: 508 (100%)
b
Tender joints counts (TJC) calculated for each patient and values averaged in each PASI group. Total ? patients: 361/508 (71%)
c
Number of nails with changes (NNC) calculated for each patient and values averaged in each PASI group. Total ? patients: 187/508 (37%)
d
AS before treatment PASI 75 versus 10
e
Doses for AS lower value: PASI 100 versus 75, 100 versus 50, 75 versus 50, 75 versus 10
f
Doses for lower tender joints values: PASI 100 versus 50, 75 versus 50, 75 versus 10
Nail changes before treatment had non-significant values similar to results obtained with tender joint counts suggesting the presence of SEC between nails and joints associated with the inflammatory process as one entity (Table 5). After 100% clinical remission of PsA and psoriasis, 15% of subjects had relapses at different times between remission and relapse. The time for remission of lesions in both diseases in the first course of treatment was similar (7.6 and 7.8 months). At relapse, PASI and AS values were two times lower than initial values before treatment. The doses for new clinical remission after relapse in both diseases were similar (7 injections), and the time needed for new remission was also similar (5.8 months) lower than the initial course of treatment (9.9 and 10.8 months) (Table 6). PBMC collected from subjects prior to treatment were analyzed by flow cytometry. LS varied with PASI range (1–10, 11–20 and 21–72) as published [42]. One group of
pre-treatment subjects had absolute values of gated LS decreased in PBMC as PASI increased. The slope decrease from high to low values was in this order: CD8?HLA-, CD8?HLA?, CD4?CD8-, CD8?CD3?, CD8?CD3(Table 7) with highest decreasing values in CD8? T cells which has been strongly implicated in PsA and might be migrating from the blood to the joints and skin as the disease gets worse. Noteworthy, the values at PASI 72 were always lower than control values (Fig. 1a). Statistical analysis with Mann–Whitney test revealed P = 0.030 between PASI 11-20 and PASI 21-72 and P = 0.041 between PASI 21–72 and control values in LS CD8?HLA-. In contrary to the previous finding, the following LS: CD8?CD4-, HLA?CD8-, CD3?CD8-, CD19?, CD8?CD4? increased in that order in PBMC. These LS had the highest positive slopes, as diseases, PsA and psoriasis got worse (Table 7), suggesting activation and
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Arch Dermatol Res Table 6 Treatment of relapse of skin psoriasis and psoriatic arthritis in 41/275 (15%) subjects, after 100% remission of lesions Initial PASI or AS
Doses for 100% remission
Time for 100% remission (months)
PASI or AS at relapse
Time from remission to relapse (months)
PASI or AS at new remission
Doses for new remission
Time for new remission (months)
Psoriasisa
19.7 ± 17.5
9.9 ± 4.8
7.6 ± 4.7
8.5 ± 11.2
12.6 ± 9.8
1.5 ± 1.9
7.8 ± 3.7
5.8 ± 3.2
PsA
15.4 ± 9.1
10.8 ± 4.7
7.8 ± 5.0
9.7 ± 6.5
17.1 ± 12.2
0.6 ± 2.0
7.2 ± 2.8
5.8 ± 3.4
AS arthritis score a
Each patient had both psoriasis and psoriatic arthritis simultaneously. Values were averaged for the 41 patients and the evolution discriminated for each disease
Table 7 Rate of change of lymphocyte subsets in peripheral blood mononuclear cells between PASI (1–10, 11–20, 21–72) in patients before treatment, and after treatment with AS100-1 Lymphocyte subsets
CD8?HLA-
Slope (S), R2, 95% confidence interval (CI) Between PASI (1–10, 11–20, 21–72) before treatment
Between 1 and 9 doses of AS100-1 after treatment
-109.9 ± 87.79, 0.610, -1,225 to 1,006
0.7195 ± 0.693, 0.1186, -0.879 to 2.318
CD8?HLA?
-53.10 ± 16.45, 0.912, -262.2 to 156.0
1.423 ± 0.5004, 0.5027, 0.269 to 2.577
CD4?CD8CD8?CD3?
-48.07 ± 33.82, 0.6690, -477.7 to 381.6 -25.40 ± 0.51, 0.999, -32.00 to -18.80
0.875 ± 1.44, 0.049, -2.547 to 4.297 1.593 ± 0.603, 0.465, 0.2007 to 2.986
CD8?CD3-
-12.15 ± 15.62, 0.377, -210.6 to 186.3
0.2633 ± 0.482, 0.0358, -0.849 to 1.376
CD8?CD4-
180.1 ± 0.011, 1.000, perfect line
0.0765 ± 1.987, 0.0002, -4.623 to 4.777
HLA?CD8-
145.1 ± 128.6, 0.559, -1,489 to 1,780
0.4259 ± 0.701, 0.044, -1.191 to 2.043
CD3?CD8-
46.85 ± 169.1, 0.0712, -2,102 to 2,196
0.986 ± 3.402, 0.013, -7.338 to 9.310
CD19?
36.25 ± 13.60, 0.876, -136.5 to 209.0
0.2494 ± 0.455, 0.0362, -0.799 to 1.298
CD8?CD4?
11.25 ± 2.742, 0.943, -23.60 to 46.10
-0.0278 ± 0.286, 0.0013,-0.705 to 0.650
IgA
143.4 ± 33.28, 0.948, -279.6 to 566.3
-0.8707 ± 0.570, 0.2495, -2.220 to 0.479
IgD
102.4 ± 79.15, 0.626, -903.3 to 1108
-0.9772 ± 0.808, 0.1727, -2.889 to 0.934
IgE
53.35 ± 1.184, 0.999, 38.31 to 68.39
0.1259 ± 0.315, 0.0222, -0.619 to 0.871
IgM
51.45 ± 2.165, 0.998, 23.94 to 78.96
0.1952 ± 0.471, 0.0238, -0.920 to 1.310
IgG
17.85 ± 4.070, 0.950, -33.87 to 69.57
0.043 ± 0.0608, 0.0666, -0.100 to 0.187
Italicize slopes are opposite and different to slopes of the same LS before treatment Statistical significant values (P \ 0.05) before treatment CD8?HLA-: PASI 11–20 versus 21–72, 21–72 versus control IgA: PASI 21–72 versus control
proliferation of T and B cells by unknown antigens at the joints and/or skin and subsequent migration to the blood. The LS values at PASI 72 were always higher or equal than control values (Fig. 1b) but with no significant statistical differences between them. Membrane surface immunoglobulin IgA?, IgD?, IgM?, IgE?, and IgG? (Table 7; Fig. 2) increased in PBMC as PASI increased, with the majority of values higher than control values, being IgA? B cells the only with statistical significant difference (P = 0.001) between PASI 21–72 and normal controls. This also suggests LS activation and proliferation by unknown antigens at joints and skin and subsequent migration to the blood (Fig. 3). After treatment with nine doses of AS100-1, the following LS: CD8?CD3?, CD8?HLA?, CD3?CD8-,
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CD4?CD8-, CD8?HLA-, HLA?CD8-, CD8?CD3-, CD19?, CD8?CD4-, CD8?CD4? had a dramatic decrease in slopes in that order as compared with the values before treatment (i.e. CD8?CD4- from S = 180.1 to S = 0.076, HLA?CD8- from S = 145.1 to S = 0.425, CD8?CD4? from S = 11.25 to S = -0.027, respectively, after treatment (Table 7). All these T and B cells have been described as responsible for the inflammatory process in joints and skin. B cells with surface immunoglobulin decreased markedly with Leishmania antigens (i.e. IgA? from S = 143.4 to S = -0.8707 and IgD? from 102.4 to S = -0.9772) exhibiting negative slopes in PBMC after treatment (Table 7; Fig. 4). One-way ANOVA between immunoglobulin groups after treatment had P B 0.0001, mean values significantly
Arch Dermatol Res Fig. 1 a Absolute values LS gated lymphocytes (average ± SEM) CD4?CD8-, CD8?HLA-, CD8?HLA?, CD8?CD3- and CD8?CD3? decreasing as PASI changes from 1–10 (A) to 11–20 (B) to 21–72 (C) units before treatment from subjects (n = 29) having 1–34 years with psoriasis and PsA, in comparison to control (D) healthy subjects (n = 35). b Absolute values LS gated lymphocytes (average ± SEM) CD8?CD4-, CD3?CD8-, HLA?CD8-, CD19?, and CD8?CD4? increasing as PASI changes from 1 to 10 (A) to 11–20 (B) to 21–72 (C) units before treatment from subjects (n = 29) having 1–34 years with psoriasis and PsA in comparison to control (D) (n = 35)
different (\0.05), R2 0.840, Tukey’s multiple comparison tests between groups significant (P \ 0.05) between IgA? versus IgD, IgA? versus IgM?, IgA? versus IgG?, IgD? versus IgE?, IgD? versus IgG?, IgM? versus IgE?, IgM? versus IgG?, IgE? versus IgG?. Two-tail unpaired t test was as follows: IgA? versus IgD? (P = 0.0001) IgA? versus IgM? (P B 0.0001), IgA? versus IgG? (P B 0.0001), IgD? versus IgE? (P B 0.0001), IgD? versus IgG? (P B 0.0001), IgM? versus IgE? (P B 0.0001), IgM? versus IgG? (P B 0.0001). The predominant decreased response with the lowest slopes was in IgA? B cells (S = -0.8707). Also, IgD? (S = -0.977) and IgG? (S = 0.043) decreased after treatment (Table 7).
Percent PASI reduction increased in time, approaching 100% reduction after nine doses of AS100-1 (S = 0.490 ± 0.163) while PASI absolute values (S = -0.065 ± 0.020), decreased after each dose of AS100-1 (Fig. 5) confirming the efficacy of Leishmania antigens in PsA.
Discussion Previous analysis of patients with all forms of psoriasis (n = 2770) from a Phase III trial exhibited PASI 100 reduction in 23% of subjects with initial PASI value 21.0 ± 1.8 [41]. The group of 508 PsA patients presented
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Arch Dermatol Res Fig. 2 Absolute values LS gated lymphocytes (average ± SEM) IgA?, IgD?, IgM?, IgE?, IgG? increasing as PASI changes from 1–10 (A) to 11–20 (B) to 21–72 (C) units before treatment grouped from subjects (n = 29) having 1–34 years with psoriasis and PsA in comparison to control (D) healthy subjects (n = 35)
here selected from the same Phase III trial but this time with more volunteers (n = 3132) had a higher percentage (54.2%) of PASI 100 reduction, with similar initial PASI (19.7 ± 17.5), since they received more doses (9.9 ± 4.8) of AS100-1 for PsA remission of lesions (Table 2) than the previous published (n = 2770) group (7.6 ± 6.0 doses) focused on skin psoriasis only [41]. All features used to define PsA clinically were more frequent in PASI 100 and PASI 75 groups with the highest number of patients and more severe diseases, as compared to PASI 50 and PASI 10. Half the total PsA study group (55.5%) independent of PASI had DIP joint involvement in hands and 16.1% in toes (Table 3). These facts were in agreement with other report describing that patients with high body surface area (BSA) currently affected by psoriasis were more likely to have developed PsA [14]. We can conclude that as more skin disease is present (higher initial PASI; Table 2), more inflammation is found in the joints (Table 3), which suggests a link between the skin disease and the inflammatory process in the joints. CD8? T cells may be activated by similar antigenic motifs in skin and joints, since CD8? LS decreased markedly in PBMC as PASI changed to 21–72 values before treatment. Furthermore, skin and joints lesions were exacerbated in the PASI 100 and PASI 75 groups and also needed higher number of doses to achieve a lower AS, tender joints and nail changes values after treatment with Leishmania antigens (Table 4). It is accepted that T lymphocytes, macrophages, antigen presenting cells (APC) and DC play a key role in PsA,
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which depends on synovial inflammation driven by unknown antigens, stimulating effector CD8? and regulatory CD4? Th-17 cells expansions. Evidence from genetic association, immunohistologic, and therapeutic studies implicates CD8? T cells in the pathogenesis of PsA [1, 3, 12]. As previously published, a vicious cycle maintains resident T cells in psoriasis [42]. B and T cells from PBMC originated in bone marrow are continuously migrating between skin and lymph nodes in a homeostatic trafficking with Langerhans cells and DC to monitor invading bacteria and virus [12, 17]. In PsA, CD8? T regulatory (Treg) might be activated by direct antigen recognition. Three subtypes of CD8? T suppressor lymphocytes (Ts) cells have been found in humans acting by: (1) transfer of inhibitory signals to APC by direct cell-to-cell contact after antigen recognition, (2) cytokine secretion without antigen activation and/or restriction, (3) stimulation by antigen recognition and induction of immunosuppressive IL-10. Once T cells and APC are activated by the unknown PsA antigen, with formation of an immunological synapse; cytokines, chemokines, and growth factors are secreted, inducing more T cell and DC activation that triggers cell proliferation at synovium [20, 21]. In the present study, we also found evidence of T cell recirculation. Before treatment, CD8?HLA-, CD8? HLA?, CD4?, CD8?CD3? and CD8?CD3- LS decreased in PBMC, while CD8?CD4-, HLA?CD8-, CD3?CD8-, CD19? and CD8?CD4? LS increased
Arch Dermatol Res Fig. 3 a Absolute values LS gated lymphocytes: CD8?HLA?, CD8?HLA-, HLA?CD8-. b LS CD4?CD8-, CD8?CD4-, CD8?CD4?, CD19; c LS CD8?CD3?, CD3?CD8-, CD8?CD3- from subjects (n = 37) after treatment with AS100-1, every 15 days
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Arch Dermatol Res Fig. 4 Absolute values LS gated lymphocytes: IgA?, IgD?, IgM?, IgE? IgG? from subjects (n = 37) after treatment with AS100-1, every 15 days
Fig. 5 % PASI reduction and PASI absolute values in PsA patients (n = 508) after treatment (1–9 doses) with AS100-1, every 15 days
(Table 7; Fig. 1a, b), following the increase in AS, tender joint counts and nail changes together with PASI values (Tables 3, 5). All mentioned T cells have been implicated in autoimmune and joints inflammatory processes [20, 21, 51]. It should be noted that in the PsA, 508 study group, CD8?HLA- was the only LS that had decreasing values with statistical significance between PASI values as PsA gets worse before treatment, in accordance with the finding by others that the majority of PsA synovial fluid CD8? T cells expressed HLA-DR antigen and CD45RO. CD8? Ts cells might also be induced downregulating the inflammatory process after treatment with Leishmania antigens. Psoriatic synovium may be related to a unique innate immune response in psoriatic skin, with similar pathologic mechanisms of connective tissue injury and innate immune activation of endogenous ligands leading to the characteristics of PsA [3, 7, 59].
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T cell proliferation was found after stimulating PBMC from psoriatic patients in a blastogenic assay with Leishmania antigens [40] a fact contrary to the majority of treatments used today that are immunosuppressors with very high side effects [49]. The management of PsA should simultaneously target arthritis, skin disease, and other manifestations of PsA, including involvement of the axial skeleton dactylitis, enthesitis, and eye inflammation. In this respect, targeted biological agents, primarily TNFa inhibitors, have emerged as generally well tolerated and highly effective alternatives to traditional ‘‘disease modifying anti-rheumatic drugs’’ (DMARDs). Although they can have some beneficial effect on skin disease and peripheral arthritis, there is lack of evidence for DMARDs, such as methotrexate (MTX), leflunomide, cyclosporine (CsA), and sulfasalazine in affecting dactylitis or enthesitis, and they
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are clearly ineffective in axial disease, furthermore they are immunosuppressors with severe SE [7, 34, 35, 63]. In a similar way, results for DMARDs response, for tender and swollen joint counts, early morning stiffness, pain visual analogue score, patient global assessment, C reactive protein (CRP) and HAQ were collected. Remission, defined according to the disease activity score using 28 joint count and CRP (DAS28-CRP), was achieved in 58% of PsA patients at 12 months [49]. By the contrary, our treatment with Leishmania amastigote antigens induced PASI 100% and PASI 75% reduction in 77.1% of patients with PsA without any SE (Table 2) since they are immunostimulators, not immunosuppressors [40]. A dramatic decrease in arthritis score, tender joints counts and number with nail changes from base line pretreatment values was obtained; even four patients in wheel chairs walked again after treatment (Table 5). After 100% clinical remission of PsA and psoriasis, 15% of subjects had relapses but PASI and AS values were two times lower than initial values. The doses for new clinical remission after relapse in both diseases were similar and lower than the initial course of treatment (Table 6) results better than with the current treatments. Traditional systemic therapies for psoriasis, such as MTX, CsA, retinoids or psoralen plus ultraviolet light (PUVA) therapy, have a potential for long-term toxicity and may not always provide sufficient improvement of the disease. Biological therapies for the treatment of PsA are defined by their mode of action and can be classified into three categories: the T cell modulating agents (alefacept and efalizumab), the TNFa blockers (adalimumab, certolizumab, etanercept, golimumab and infliximab) and the inhibitors of interleukin IL-12 and IL-23 (ustekinumab and briakinumab) [50, 54, 60]. Despite the use of these treatments, 30–40% of patients will still have active disease [49]. This is in contrast to 90% reduction in AS, TJC and NNC values as compared to baseline values in Leishmania antigen-treated patients (Table 5). DTH-positive reactions were found with isolated amastigote DEAE fractions in humans in vivo, after treatment with AS100-1 [41]. These facts of in vivo and in vitro T cells stimulation suggest that variations in blood LS, before treatment at different PASI values in PsA subjects, are function of lymphocyte migration from blood to joints and skin and vice versa, supported by T cell activation in synovium and skin plaques as the disease gets worse. On the other hand, after treatment, T and B cells are not killed by Leishmania antigens [40]; they are probably deactivated or neutralized by CD8? T reg and/or T suppressor cells, decreasing the vicious cycle or cell recirculation between, joints and/or skin and blood. In normal subjects, 2% of PBMC express both CD4 and CD8 molecules on the surface of large granular blast
lymphocytes in PBMC. Double positive (DP) CD4?CD8? T cells are an activated phenotype of CD8? T cells, expressing high levels of CD2, CD29, CD56, and CD57. In addition, the CD4?CD8? cells coexpressed CD45RA and CD45RO suggesting an intermediate state between naive and memory T cells [51]. Our present data on PsA before treatment suggest that stimulation by an unknown antigen induces DP CD8?CD4? LS in synovium and skin as well as the activated DP phenotypes CD8?HLA? and CD8?CD3? LS by sensitization of CD8? T cells (Table 7). It is noteworthy that Leishmania antigens bind CD2 receptors in PBMC from normal healthy subjects never exposed to Leishmania infection, which is present in CD4?CD8? cells [29]. Thus, we postulate that before treatment, DP cells are activated in the lymph-node-like structure in the inflamed joint and/or skin, or in draining lymph nodes and recirculate back to the blood via lymph node lymphatic vessels as single positive (SP) CD8?CD4-, HLA?CD8-, CD3?CD8-, CD19? and DP CD8?CD4? LS (positive slopes in PBMC; Table 7) as PASI and AS score approach higher values. Subsequently they enter the skin or joints in high numbers as SP or DP CD8?HLA-, CD8?HLA?, CD4?, CD8?CD3?CD8?CD3-, T cells decreasing in PBMC (negative slopes as PASI and AS increases; Table 7) as PsA gets worse, contributing to the vicious cycle described above. It is noteworthy that the highest LS decreased as measured by negative slopes had the CD8? phenotype postulated as responsible for PsA inflammation in joints [12, 17, 57]. After AS100-1 vaccine, all LS in PBMC decreased markedly in slope values, the inverse result to the observed situation before treatment. A dramatic decrease in DP activated cells in PBMC: CD8?HLA? (S = 0.77), CD8?CD4? (S = 0.211), CD8?CD3? (S = 1.962), and SP cells: CD8?CD3- (S = 1.015) CD8?HLA(S = 1.338) was observed (Table 7). Thus, these LS cannot repopulate the skin and joints after treatment because their low concentration in PBMC which paralleled the reduction in AS, tender joint counts, nail changes (Table 4) and PASI values (Fig. 5). As a result the vicious cycle described above disappears. DP CD8?CD3? T cells consists of 0.2–7.0% of all CD8 T cells in healthy donors, and represent a subset of activated CD8? effector T cells, resulting from a continuous immune response to intracellular pathogens [57]. In our work we used intracellular particulate membrane antigens from Leishmania amastigote parasites. SP and DP CD8? LS subsets decreased in PBMC after nine doses of AS100-1, probably by decreasing proliferation in psoriatic plaques and/or joint synovium, due to desensitization after binding CD2 receptor [29] in the immunological synapse [40–42] or induction of T regulatory cells. Treg alterations
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Arch Dermatol Res
in the peripheral blood may correlate with normal or increased function at the site of inflammation, likely suggesting the occurrence of recirculation of these cells and homing to inflamed tissues, as published [20, 21]. The increase in IgA?, IgD?, IgM? IgE? IgG? cells as PASI increased (Fig. 2) and the significant decrease after treatment with AS100-1 (Table 7; Fig. 4) suggests deactivation of B cells, and confirms recirculation between blood, skin and joints as found in our work and makes it possible to investigate the antigens sensitizing B cells and their role in the pathogenesis of PsA. PsA has been associated with a heat shock protein-like transmembranous protein MICA, a ligand for several immune cells and NK cells. MICA-A9 appears to be the strongest genetic susceptibility factor for PsA. MICA is a membrane protein that is mainly expressed in epithelial cells of the intestine and is recognized by NKG2D receptor expressed on NK cells, CD8ab T cells, and cd T lymphocytes and plays a role in the innate defense against a biological stress [13, 25]. In our work, intracellular amastigote antigens are isolated from Leishmania parasites, after a heat shock induced in promastigotes, which became transformed into intracellular amastigote forms [43]. It is conceivable that heat shock proteins (Hsp) from amastigotes are reacting with MICA, deactivating those cells and down regulating the inflammatory process in PsA. Interestingly, the molecular weights of the fractions that induce clinical remission of psoriasis are between 59 and 65 kDa in the range of Hsp60, as published previously [40, 41]. Purified protein antigens from L(V) brasiliensis AS200 had an antiarthritic effect in a CIA model. AS100-1 also decreased the effect of CIA in forepaws [39], as found in humans with AS100-1 in the present work Hsp are highly conserved and immunogenic proteins shared by microbial agents and mammals. Long-term Hsp confrontation of the immune system similar in the host and invaders may convert the immune response against these host antigens and promote and/or decrease autoimmune diseases including RA, atherosclerosis, type 1 diabetes and psoriasis. Hsp also possess immunoregulatory attributes as well and therefore could be used as immunomodulators for immune-mediated disorders [45, 64]. There is evidence that recognition of self-Hsp60 can have beneficial effects in arthritis. Control measures in the inflammatory processes can be achieved by administration of peptides cross-reactive to self-determinants [59, 61, 62]. Amastigote peptides produced after a heat shock in promastigotes might be cross reacting with host Hsp, immunomodulating the response in PsA and psoriasis, thereby decreasing the inflammatory process in synovium and skin as found in our work. Th-17 T cells might also be downregulated by amastigote peptides, since we obtained remission of forepaw lesions in the CIA model in mice. The aluminum compounds were originally identified as adjuvants over 70 years ago and have been commonly used
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as an adjuvant in many vaccines licensed by the US Food and Drug Administration. The requirement for association of antigen with particulate adjuvants is well recognized, and it has been proposed that the ability of particulates to target antigen to phagocytes may underlie their adjuvant activity [4]. In our case we have demonstrated that Leishmania antigens absorbed to alumina induced cellular immunity [40, 41]. Groups of BALB/c mice were immunized with L. major exogenous antigens (SEAgs) alone, L. major SEAgs coadministered with either aluminum hydroxide or recombinant murine interleukin-12 (rmIL-12), L. major SEAgs coadministered with both alum and rmIL-12, and L. major SEAgs coadministered with Montanide ISA 720. The adjuvant alone had no effect. Importantly and surprisingly, the greatest and most consistent protection against challenge with L. major was seen in mice immunized with L. major SEAgs alone, in the absence of any adjuvant [56]. In the mice ConA-induced hepatitis model, Rehydragel placebo had no effect as compared to dexamethasone, or Leishmania antigenic proteins in decreasing IL-1b inflammatory marker in mice blood as published [38]. Aluminum hydroxide directly stimulates monocytes to produce proinflammatory cytokines activating T cells. Activated Th2 cells release IL-4, which, in turn, can induce an increase in the expression of MHC class II molecules on monocytes in vitro, which may explain its potent in vivo adjuvant effect [58]. Intramuscular administration of aluminum-adjuvanted vaccines induced an infiltration of aluminum-containing macrophages between muscle fibers in mice and humans. In vitro stimulation of human monocyte-derived macrophages with aluminum hydroxide induces similar intracellular crystalline inclusions as well as phenotypical and functional modifications characterized by autologous memory T lymphocyte proliferation, CD83 expression and increased CD86 on macrophages which may also explain the adjuvant effects [47, 48]. Toll-like receptor-2 (TLR2) binds Gram-positive bacterial cell wall proteins such as peptidoglycans and bacterial lipopeptides. Stimulation of TLR2 in rheumatoid arthritis synovial fibroblasts (RASF) in the innate immune system upregulates the production of pro-inflammatory cytokines IL-6 and IL-15, chemokines, as well as matrix degrading metalloproteinase, adhesion molecules and subsequent osteoclastogenesis [31]. Heat shock proteins Hsp60, Hsp70, Gp96 function as host-derived ligands for TLR2, and play a role in the pathogenesis of RA and psoriasis. Hsp60 is a mitochondrial chaperonin that is involved in the pathogenesis and immunomodulation of autoimmune diseases [45]. It should be noted that Leishmania have big mitochondria all around the cytoplasm in promastigotes and amastigotes parasites a likely source of Hsp. The molecular weight of AS100-1 had bands in the
Arch Dermatol Res
range of most Hsp host ligands (50–70 kDa) for TLR2 and could be competing with peptides in RASF TLR2 receptors downregulating pro-inflammatory cytokines inhibiting the inflammation in PsA. TNFa plays a central role in mediating inflammation in PsA [1]. C reactive protein (CRP) and complement factor C5a (C5a) decreased markedly in serum after six doses of AS200 Leishmania antigens in psoriatic patients [38]. TNFa, also decreased in PBMC supernatants of patients after Concanavalin A (ConA) stimulation in the presence of AS100-2 monovalent second generation L(L) chagasi antigens [38]. In addition, TNFa was found to decrease in serum of mice with ConA-induced hepatitis after injection of AS100-2 L(L) chagasi antigens [38]. This suggests that Leishmania amastigote peptides are blocking the ConA receptor both in vitro and in vivo downregulating TNFa secretion. Similar results were obtained in the mice ConA model with IL-1b serum concentration after 8 h of injection of dexamethasone, AS200, and AS100-1 Leishmania antigens [38]. These experiments suggest that immunization with Leishmania amastigote antigens decreases inflammatory markers and inflammatory cytokines in PsA and psoriasis inducing regression of joint and skin lesions. The immune response to AS100-1 was cellular in nature and not humoral as supported by the DTH and ELISA results in humans [40, 41] and DTH response in guinea pigs [39]. All psoriatic patients were DTH positive after the third vaccination with AS100-1, but no ELISA antibodies were detected in serum from these volunteers up to six doses of vaccine [41]. It seems plausible that the immunological response to AS100-1 after amastigotes TLCK treatment and NP-40 extraction was truly a cellular response by immunization of several kinds of T cells, including T-regulatory cells, with no antibody production, a novel mechanism that may play a role in decreasing inflammation in PsA joints and psoriatic skin. Amastigote peptides may induce CD8? regulatory T cells producing IL-10 that inhibits Th1 and Th2 cell cytokine production and induces peripheral cell tolerance [21]. Finally, TNF has been implicated in many diseases such as Crohn’s disease, multiple sclerosis, Alzheimer’s disease, transplant rejection, type II diabetes, rheumatoid arthritis, heart failure, atherosclerosis, allergic asthma, liver disease, tumorigenesis, tumor metastasis, lymphoproliferative diseases, pulmonary fibrosis, and systemic lupus erythematosus. Most of these diseases have animal models in which the efficacy of Leishmania amastigote antigens could be examined.
Conclusion As more skin disease is present in PsA patients, more inflammation is found in the joints, suggesting a link
between skin and joint inflammatory processes; since both were exacerbated in the PASI 100 and PASI 75 groups and also needed higher number of doses to achieve a lower AS, tender joints and nail changes values. Absolute values of gated LS before treatment decreased in this order: CD8?HLA-, CD8?HLA?, CD4?, CD8?CD3?, CD8? CD3 in PBMC as PASI increased, suggesting migration of CD8? cells from the blood to the joints and skin. In contrary to the previous finding, LS: CD8?CD4-, CD3?CD8-, HLA?CD8-, CD19?, CD8?CD4?, IgA?, IgD?, IgM?, IgE?, and IgG? increased in PBMC as PASI increased suggesting activation and proliferation by unknown antigens. There is evidence of T cell recirculation before treatment and a vicious cycle with T and B cells migrating between blood and skin and joints. After treatment with AS100-1 Leishmania antigens, a dramatic decrease in all T and B LS in PBMC was observed, as PASI, AS, tender joint counts, and nail changes returns to normal values and the vicious cycle disappears. Ethic commission The clinical investigations were conducted in accordance with the Declaration of Helsinki. The Ethic Commission of the National Academy of Medicine of Venezuela approved the protocols for the field trial for leishmaniasis as well as the trials for psoriasis and psoriatic arthritis. Dr. Blas Bruni Celli was appointed to trial monitor and oversaw all subsequent follow-up work on the trials. All volunteers signed an informed consent authorizing treatment. Acknowledgments Jose A. O’Daly, Astralis CSO, CEO and Chairman, would also like to thank The Technology Business Tax Certificate Transfer Program of the Greater State of New Jersey, Economic Development Authority (eda) and to the Consejo Nacional de Investigaciones Cientificas y Tecnologicas (CONICIT) for its support, under contract # RP-IV-120030, while Dr. O’Daly was in Caracas, Venezuela.
References 1. Amherd-Hoekstra A, Na¨her H, Lorenz HM et al (2010) Psoriatic arthritis: a review. J Dtsch Dermatol Ges 8:332–339 2. Barton AC (2002) Genetic epidemiology. Psoriatic arthritis. Arthritis Res 4:247–251 3. Boyle DL, Kavanaugh A (2008) The pathobiology of psoriatic synovium. Curr Opin Rheumatol 20:404–407 4. Brewer JM (2006) (How) do aluminium adjuvants work? Immunol Lett 102:10–15 5. Brockbank JE, Stein M, Schentag CT et al (2005) Dactylitis in psoriatic arthritis: a marker for disease severity? Ann Rheum Dis 64:188–190 6. Cassell SE, Bieber JD, Rich P et al (2007) The modified nail psoriasis severity index: validation of an instrument to assess psoriatic nail involvement in patients with psoriatic arthritis. J Rheumatol 2007 34:123–129
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Arch Dermatol Res 7. Ceponis A, Kavanaugh A (2010) Treatment of psoriatic arthritis with biological agents. Semin Cutan Med Surg 29:56–62 8. Chandran V, Gottlieb A, Cook RJ et al (2009) Psoriatic arthritis international multicenter psoriasis and psoriatic arthritis reliability trial for the assessment of skin, joints, nails, and dactylitis. Arthritis Rheum 61:1235–1242 9. Chandran V, Raychaudhuri SP (2010) Geoepidemiology and environmental factors of psoriasis and psoriatic arthritis. J Autoimmun 34:314–321 10. Chandran V, Schentag CT, Gladman DD (2007) Sensitivity of the classification of psoriatic arthritis criteria in early psoriatic arthritis. Arthritis Rheum 57:1560–1563 11. Chang YT, Chen TJ, Liu PC et al (2009) Epidemiological study of psoriasis in the national health insurance database in Taiwan. Acta Derm Venereol 89:262–266 12. Choy E (2007) T cells in psoriatic arthritis. Curr Rheumatol Rep 9:437–441 13. Choy MK, Phipps ME (2010) MICA polymorphism: biology and importance in immunity and disease. Trends Mol Med 16:97–106 14. Christophers E, Barker JN, Griffiths CE et al (2010) The risk of psoriatic arthritis remains constant following initial diagnosis of psoriasis among patients seen in European dermatology clinics. J Eur Acad Dermatol Venereol 24:548–554 15. Ciocon DH, Kimball AB (2007) Psoriasis and psoriatic arthritis: separate or one and the same? Br J Dermatol 157:850–860 16. Coates LC, Helliwell PS (2008) Classification and categorization of psoriatic arthritis. Clin Rheumatol 27:1211–1216 17. Costello P, Bresnihan B, O’Farrelly C et al (1999) Predominance of CD8? T lymphocytes in psoriatic arthritis. J Rheumatol 26:1117–1124 18. Elder JT (2009) Genome-wide association scan yields new insights into the immunopathogenesis of psoriasis. Genes Immun 10:201–209 19. Englbrecht M, Wang Y, Ronneberger M et al (2010) Measuring joint involvement in polyarticular psoriatic arthritis: an introduction of alternatives. Arthritis Care Res 62:977–983 20. Filaci G, Fenoglio D, Indiveri F (2011) CD8(?) T regulatory/ suppressor cells and their relationships with autoreactivity and autoimmunity. Autoimmunity 44:51–57 21. Filaci G, Rizzi M, Setti M et al (2005) Non-antigen-specific CD8(?) T suppressor lymphocytes in diseases characterized by chronic immune responses and inflammation. Ann N Y Acad Sci 1050:115–123 22. Fink AM, Cauza E, Hassfeld W et al (2007) Vascular endothelial growth factor in patients with psoriatic arthritis. Clin Exp Rheumatol 25:305–308 23. FitzGerald O, Winchester R (2009) Psoriatic arthritis: from pathogenesis to therapy. Arthritis Res Ther 11:214–223 24. Gabriel SE, Michaud K (2009) Epidemiological studies in incidence, prevalence, mortality, and comorbidity of the rheumatic diseases. Arthritis Res Ther 11:229–245 25. Gonza´lez S, Martı´nez-Borra J, Lo´pez-Va´zquez A et al (2002) MICA rather than MICB, TNFA, or HLA-DRB1 is associated with susceptibility to psoriatic arthritis. J Rheumatol 29:973–978 26. Griffiths EMG, Barker JN (2007) Pathogenesis and clinical features of psoriasis. Lancet 370:263–271 27. Ibrahim G, Waxman R, Helliwell PS (2009) The prevalence of psoriatic arthritis in people with psoriasis. Arthritis Rheum 61:1373–1378 28. Jandus C, Bioley G, Rivals JP et al (2008) Increased numbers of circulating polyfunctional Th17 memory cells in patients with seronegative spondylarthritides. Arthritis Rheum 58:2307–2317 29. Kemp M, Hansen MB, Theander TG (1992) Recognition of Leishmania antigens by T lymphocytes from nonexposed individuals. Infect Immun 60:2246–2251
123
30. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193(1):265–275 31. Maciejewska H, Ju¨ngel A, Gay RE et al (2009) Innate immunity, epigenetics and autoimmunity in rheumatoid arthritis. Mol Immunol 47:12–18 32. McGonagle D, Lories RJU, Tan AL et al (2007) The concept of a ‘‘synovio-entheseal complex’’ and its implications for understanding joint inflammation and damage in psoriatic arthritis and beyond. Arthritis Rheum 56:2482–2491 33. McGonagle D (2009) Enthesitis: an autoinflammatory lesion linking nail and joint involvement in psoriatic disease. J Eur Acad Dermatol Venereol 23:9–13 34. Mease P (2006) Psoriatic arthritis update. Bull NYU Hosp Jt Dis 64:25–31 35. Mease PJ (2010) Psoriatic arthritis: pharmacotherapy update. Curr Rheumatol Rep 12:272–280 36. Mrowietz U, Reich K (2009) Psoriasis—new insights into pathogenesis and treatment. Dtsch Arztebl Int 106:11–19 37. Mrowietz U, Elder JT, Barker J (2007) The importance of disease associations and concomitant therapy for the long term management of psoriasis patients. Arch Dermatol Res 298:309–319 38. O’Daly JA, Gleason J (2010) Antigens from Leishmania amastigotes inducing clinical remission of psoriasis: relationship between leishmaniasis and psoriasis. DERMA 1:47–57 39. O’Daly JA, Gleason JP, Pen˜a G et al (2010) Purified proteins from leishmania amastigotes-induced delayed type hypersensitivity reactions and remission of collagen-induced arthritis in animal models. Arch Dermatol Res 302:567–581 40. O’Daly JA, Lezama R, Gleason J (2009) Isolation of Leishmania amastigote protein fractions which induced lymphocyte stimulation and remission of psoriasis. Arch Dermatol Res 301:411–427 41. O’Daly JA, Lezama R, Rodriguez PJ et al (2009) Antigens from Leishmania amastigotes induced clinical remission of psoriasis. Arch Dermatol Res 301:1–13 42. O’Daly JA, Rodriguez B, Ovalles T et al (2010) Lymphocyte subsets in peripheral blood of patients with psoriasis before and after treatment with leishmania antigens. Arch Dermatol Res 302:95–104 43. O’Daly JA, Rodriguez MB (1988) Differential growth requirements of several leishmania spp in chemically defined culture media. Acta Trop Basel 45:109–126 44. Radtke MA, Reich K, Blome C et al (2009) Prevalence and clinical features of psoriatic arthritis and joint complaints in patients with psoriasis: results of a German national survey. J Eur Acad Dermatol Venereol 23:683–691 45. Rajesh R, Moudgil KD (2009) Heat-shock proteins can promote as well as regulate autoimmunity. Autoimmun Rev 8:388–393 46. Reich K, Kruger K, Mossner R et al (2009) Epidemiology and clinical pattern of psoriatic arthritis in Germany: a prospective interdisciplinary epidemiological study of 1511 patients with plaque-type psoriasis. Br J Dermatol 160:1040–1047 47. Rimaniol AC, Gras G, Clayette P (2007) In vitro interactions between macrophages and aluminum-containing adjuvants. Vaccine 25:6784–6792 48. Rimaniol AC, Gras G, Verdier F et al (2004) Aluminum hydroxide adjuvant induces macrophage differentiation towards a specialized antigen-presenting cell type. Vaccine 22:3127–3135 49. Rozenblit M, Lebwohl M (2009) New biologics for psoriasis and psoriatic arthritis. Dermatol Ther 22:56–60 50. Saber TP, Ng CT, Renard G et al (2010) Remission in psoriatic arthritis: is it possible and how can it be predicted? Arthritis Res Ther 12:R94 51. Sala P, Tonutti E, Feruglio C et al (1993) Persistent expansions of CD4? CD8? peripheral blood T cells. Blood 82:1546–1552
Arch Dermatol Res 52. Salliot C, Dernis E, Lavie F et al (2009) Diagnosis of peripheral psoriatic arthritis: recommendations for clinical practice based on data from the literature and experts opinion. Joint Bone Spine Joint Bone Spine 76:532–539 53. Smith PK, Krohn RI, Hermanson GT et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85 54. Soriano ER, Rosa J (2009) Update on the treatment of peripheral arthritis in psoriatic arthritis. Curr Rheumatol Rep 11:270– 277 55. Taylor W, Gladman D, Helliwell P et al (2006) Classification criteria for psoriatic arthritis: development of new criteria from a large international study. Arthritis Rheum 54:2665–2673 56. Tonui WK, Mejia JS, Hochberg L et al (2004) Immunization with Leishmania major exogenous antigens protects susceptible BALB/c mice against challenge infection with L major. Infect Immun 72:5654–5661 57. Trautmann A, Ru¨ckert B, Schmid-Grendelmeier P et al (2003) Human CD8 T cells of the peripheral blood contain a low CD8 expressing cytotoxic/effector subpopulation. Immunology 108:305–312 58. Ulanova M, Tarkowski A, Hahn-Zoric M et al (2001) The common vaccine adjuvant aluminum hydroxide up-regulates
59.
60.
61.
62.
63. 64.
accessory properties of human monocytes via an interleukin-4dependent mechanism. Infect Immun 69:1151–1159 van Kuijk AW, Reinders-Blankert P, Smeets TJ et al (2006) Detailed analysis of the cell infiltrate and the expression of mediators of synovial inflammation and joint destruction in the synovium of patients with psoriatic arthritis: implications for treatment. Ann Rheum Dis 65:1551–1557 Weger W (2010) Current status and new developments in the treatment of psoriasis and psoriatic arthritis with biological agents. Br J Pharmacol 160:810–820 Winchester R, Minevich G, Kane D et al (2008) Heterogeneity of the psoriasis phenotype revealed by HLA class I haplotype associations in psoriatic arthritis and psoriasis. Clin Immunol 127:S88–S89 Winchester R (2004) The genetics of autoimmune-mediated rheumatic diseases: clinical and biologic implications. Rheum Dis Clin North Am 30:213–227 Wollina U, Unger L, Heinig B, Kittner T (2010) Psoriatic arthritis. Dermatol Ther 23:123–136 Zu¨gel U, Kaufmann SHE (1999) Role of heat shock proteins in protection from and pathogenesis of infectious diseases. Clin Microbiol Rev 12:19–39
123
