Efficacy and safety of HAT1 compared with calcipotriol in the treatment of patients with mild to moderate chronic plaque psoriasis: results from an open-label randomized comparative pilot clinical study.

Psoriasis is commonly treated with topical corticosteroids, oral cytotoxic drugs and biologic agents, which can be associated with significant adverse effects (AEs), high cost and response attenuation. Additionally, patients often use alternative therapies ad hoc, which can be challenging to integrate into a treatment regimen, owing to a lack of adequately powered controlled trials assessing efficacy and safety. We developed a novel topical botanical complex, herbal anti-inflammatory treatment (HAT1), through extensive preclinical in vitro and in vivo modelling to define key mechanisms of action and clinical potential. To assess the efficacy and safety of HAT1 in psoriasis, we performed a 10-week, open-label, pilot clinical trial comparing topical treatment of HAT1 with calcipotriol 0.005% in adult patients with mild to moderate psoriasis. Primary and secondary endpoints included the percentage of patients obtaining improvement of ≥ 75% in Psoriasis Area and Severity Index (PASI 75), Physician's Global Assessment (PGA) response, and evaluation of tolerability and safety of HAT1. In the HAT1 arm, 85.7% of study patients reached PASI 75 compared with 21.4% in the calcipotriol comparator group. Additionally, 78.6% of patients in the HAT1 arm achieved a 'clear' or 'minimal' PGA response. HAT1 was well tolerated, with no AEs observed throughout the trial. These results suggest that HAT1 reduces psoriasis disease activity in a clinically relevant manner. Ongoing studies, including well-powered, double-blind, randomized controlled trials will be required to assess the potential of HAT1 in psoriasis.

Advances in below-the-knee drug-eluting balloons.

The management of critical limb ischemia due to below-the-knee disease remains challenging due to the frequent patient comorbidities, diffuse vascular involvement, and high rates of restenosis and disease progression. The BASIL study has established the substantial equivalence between bypass surgery and percutaneous transluminal angioplasty in this setting, at least at mid-term follow-up, but percutaneous techniques and devices have seen major developments since the publication of this pivotal trial in 2005. A major breakthrough has indeed been the introduction of drug-eluting balloons, which have several theoretical advantages in comparison to standard balloons and metallic stents for infra-popliteal lesions. Two clinical trials have already been reported with favorable results for the In.Pact Amphirion paclitaxel-eluting balloon, when employed for below-the-knee lesions. We hereby discuss the rationale for the use of drug-eluting balloons in this complex setting and the main findings of the study by Schmidt et al. and the DEBATE-BTK trial.

Apolipoprotein-defined lipoprotein abnormalities in rheumatoid arthritis patients and their potential impact on cardiovascular disease.

OBJECTIVES: The aim of this study was to explore apolipoprotein-defined lipoproteins for abnormalities when comparing non-rheumatological controls to rheumatoid arthritis (RA) patients. METHODS: Apolipoprotein and lipoprotein profiles were measured on 94 RA patients and 79 controls by immunoturbidimetric procedures, electroimmunoassays, and immunoprecipitation. Differences between means were tested with a two-sided Student t test with Satterthwaite adjustment. p-values were adjusted for multiple comparisons using the Bonferroni procedure. RESULTS: RA patients had significantly higher levels of total cholesterol (TC), triglycerides (TG), and very low density lipoprotein cholesterol (VLDL-C) than controls, but no significant differences in the levels of high density lipoprotein cholesterol (HDL-C) and LDL-C. RA patients had significantly lower levels of apolipoprotein (apo)A-I and lipoprotein (Lp)A-I:A-II, but no difference in levels of LpA-I than normal controls. There was a significant difference in the levels of LpB:C but not in LpB:C:E between RA patients and controls. The main abnormality among apoB lipoproteins was the significantly increased concentration of the LpA-II:B:C:D:E subclass in RA patients in comparison with controls. The high levels of LpA-II:B:C:D:E are also reflected in significantly increased levels of apoC-III, and apoC-III bound to apoB lipoproteins. CONCLUSION: The LpA-II:B:C:D:E subclass has potential as a new marker for cardiovascular disease (CVD) in RA patients.

Apolipoprotein B-containing lipoprotein subclasses as risk factors for cardiovascular disease in patients with rheumatoid arthritis.

OBJECTIVE: The purpose of this study was to explore whether nontraditional risk factors, such as apolipoprotein C-III (Apo C-III) and its corresponding Apo B lipoprotein (Lp) subclasses, contribute to the risk of cardiovascular disease in rheumatoid arthritis (RA) patients. METHODS: Apolipoprotein and lipoproteins were measured in 152 RA patients by immunoturbidimetric procedures, electroimmunoassay, and immunoprecipitation. Patients had a coronary artery calcium (CAC) score assessed at baseline and at year 3. Differences in the CAC scores between baseline and year 3 were calculated and dichotomized at 0, where patients with a difference score >0 were denoted as progressors and the rest were denoted as nonprogressors. Differences between means were tested with a 2-sided independent Student's t-test with Satterthwaite's adjustment. Proportion differences were tested with a chi-square test. Multiple logistic regression was performed to assess the relationship between apolipoprotein and lipoprotein levels and the dichotomized CAC score. RESULTS: Progressors accounted for almost 60% of the cohort. Progressors had significantly higher levels of triglycerides, very low-density lipoprotein (VLDL) cholesterol, total cholesterol/high-density lipoprotein (HDL), triglycerides/HDL, Apo B, LpA-II:B:C:D:E, LpB:C, Apo B/Apo A-I, Apo C-III, and Apo C-III-heparin precipitate than the nonprogressors. After adjusting for age, sex, statin use (yes/no), and hypertension (yes/no), significant risk factors of progressors were total cholesterol, triglycerides, VLDL cholesterol, LDL cholesterol, Apo B, LpB:C, Apo C-III, and Apo B/Apo A-I. CONCLUSION: Apo C-III-containing Apo B lipoprotein subclasses were found to be significantly elevated in progressors compared to nonprogressors. Many of these same lipoproteins were found to be associated with an increase in CAC scores among progressors. These lipoproteins may be considered new risk factors for progression of atherosclerosis in RA patients.

Angioplasty of below-the-elbow arteries in critical hand ischaemia.

BACKGROUND: Critical hand ischaemia (CHI) due to pure below-the-elbow (BTE) artery obstruction is a disabling disease and there is still no consensus concerning the most appropriate revascularisation strategy. The aim of this study was to assess the feasibility, safety and outcomes of percutaneous transluminal angioplasty (PTA) in the treatment of CHI due to pure BTE artery disease. METHODS AND RESULTS: Twenty-eight patients (age 62 ± 11 years; three females) with a total of 34 hands affected by CHI (one pain at rest; 18 non-healing ulcer; 15 gangrene) due to pure BTE artery disease underwent PTA. Most of the patients were males with a long history of diabetes mellitus, end-stage renal disease (ESRD) on haemodialysis and systemic atherosclerosis. The interosseous artery was free of disease in all cases, whereas the radial and ulnar arteries were simultaneously involved in 31/34 hands with long stenosis/occlusions (91%; mean length 155 ± 64 mm). The technical success rate was 82% (28/34), with only three minor complications. In the three cases with a functioning radial arteriovenous fistula, we successfully treated the ulnar artery. PTA was unsuccessful in 18% (6/34) hands due to inability to cross severely calcified lesions. The hand-healing rate was 65% (22/34). The predictors of hand healing were PTA technical success (odds ratio (OR) 0.5, confidence interval (CI) 0.28-0.88; p ≤ 0.0001) and digital run-off (OR 0.37, CI 0.19-0.71; p ≤ 0.003). The mean follow-up period was 13 ± 9 months. Six patients (18%) underwent secondary procedures due to symptomatic restenosis. In all these cases, a successful re-PTA was performed at a mean 6 months after the index procedure, and there were no major procedure-related events. Ten patients (36%) died during follow-up. CONCLUSIONS: Angioplasty of BTE vessels for CHI is a feasible and safe procedure with acceptable rates of technical success and hand healing. Poor digital run-off due to obstructive disease of the digital vessels can reduce the hand-healing rate after a successful PTA. Pure isolated BTE vessel disease seems to characterise patients with ESRD and diabetes mellitus.

Combining electrospinning and fused deposition modeling for the fabrication of a hybrid vascular graft.

Tissue engineering of blood vessels is a promising strategy in regenerative medicine with a broad spectrum of potential applications. However, many hurdles for tissue-engineered vascular grafts, such as poor mechanical properties, thrombogenicity and cell over-growth inside the construct, need to be overcome prior to the clinical application. To surmount these shortcomings, we developed a poly-L-lactide (PLLA)/poly-epsilon-caprolactone (PCL) scaffold releasing heparin by a combination of electrospinning and fused deposition modeling technique. PLLA/heparin scaffolds were produced by electrospinning in tubular shape and then fused deposition modeling was used to armor the tube with a single coil of PCL on the outer layer to improve mechanical properties. Scaffolds were then seeded with human mesenchymal stem cells (hMSCs) and assayed in terms of morphology, mechanical tensile strength, cell viability and differentiation. This particular scaffold design allowed the generation of both a drug delivery system amenable to surmount thrombogenic issues and a microenvironment able to induce endothelial differentiation. At the same time, the PCL external coiling improved mechanical resistance of the microfibrous scaffold. By the combination of two notable techniques in biofabrication--electrospinning and FDM--and exploiting the biological effects of heparin, we developed an ad hoc differentiating device for hMSCs seeding, able to induce differentiation into vascular endothelium.

Ablation of Stat3 by siRNA alters gene expression profiles in JEG-3 cells: a systems biology approach.

Control of inflammation at the maternal-fetal interface is a critical element in mammalian pregnancy. Previous work from our laboratory has shown that Stat3 may be a placental mediator involved in maintaining immunologic homeostasis at the maternal-fetal interface. The aim of the current study is to further elucidate the role of Stat3 in response to inflammation. As ablation of Stat3 in mice results in embryonic lethality, we evaluated the role of Stat3 in vitro using an siRNA approach. Trophoblast-like JEG-3 cells were transfected with an siRNA construct specific to Stat3. Experimental and control cells were exposed to conditioned medium from PHA-activated peripheral blood mononuclear cells and incubated for 45 min. Cells were then collected and RNA isolated for transcriptional profiling using human Affymetrix U133 plus 2.0 GeneChips. Differences in gene expression between control and Stat3-ablated cells were evaluated using conventional statistical methods. Fifty-two genes were detected as up-regulated in conditioned medium in both mock transfected and in Stat3 siRNA transfected JEG-3 cells. Two genes (EPAS1 and RASGEF1B) were up-regulated only in cells transfected with negative control siRNA, while 36 genes were up-regulated only in cells transfected with Stat3 siRNA. Sixty genes were differentially expressed between Stat3 siRNA transfected cells relative to mock transfected cells both in basal and conditioned medium. These included 31 genes up-regulated with Stat3 siRNA transfected cells and 29 genes down-regulated with Stat3 siRNA. Eleven genes were differentially expressed only in basal medium. Seven of these were up-regulated in the presence of Stat3 siRNA and four were down-regulated. Nine genes were differentially expressed only in conditioned medium. Six of these were up-regulated and three down-regulated in the presence of Stat3 siRNA. Off-target effects were excluded in a second set of experiments in which Stat3 mRNA was targeted at a different site and quantitative real-time PCR performed on selected genes derived from the microarray analysis. While some of the genes that showed differential expression between Stat3-ablated cells and mock transfected controls were genes typically associated with immune response (e.g., CCR7 and IRAK1), in silico modeling of the microarray data also revealed complex networks of signaling molecules and molecules associated with cellular metabolism previously seen in transcription factor ablation in model organisms. We conclude thus: Stat3 controls a specific gene set in trophoblast-like JEG-3 cells. While some differentially expressed genes and in silico models of their functions are consistent with the hypothesis that Stat3 plays a role in regulating inflammation, Stat3-mediated response to inflammation appears to also involve complex homeostatic adaptations of a non-immunologic nature.

Long-term outcomes after angioplasty of isolated, below-the-knee arteries in diabetic patients with critical limb ischaemia.

BACKGROUND: It has been shown that concomitant percutaneous transluminal angioplasty (PTA) of above-the-knee (ATK) and below-the-knee (BTK) arteries is highly beneficial for limb salvage in patients with critical limb ischaemia (CLI), but few published studies have specifically investigated outcomes in diabetic patients with CLI associated with isolated small BTK-vessel disease. This study aimed to evaluate the long-term results of successful PTA for limb salvage in such patients. MATERIALS AND METHODS: From among the 634 patients with CLI in our database, we retrospectively selected a consecutive series of 101 diabetics (16%) with 107 critically ischaemic limbs (33 Rutherford 5 and 74 Rutherford 6) and no critical ATK lesion, who underwent PTA on isolated BTK lesions. RESULTS: The limb salvage rate was 93% after a mean follow-up of 1048+/-525 days (2.9+/-1.4 years). Transcutaneous oxygen tension significantly increased after 1 month (18.1+/-11.2 vs. 39.6+/-15.1; p<0.05). After 1 year, target-vessel re-stenosis had occurred in 42% of the non-amputated limbs, nine patients (9%) had died because of medical conditions unrelated to PTA and three patients had undergone repeat PTA for recurrent CLI. CONCLUSIONS: In our selected patient population with ischaemic diabetic foot and isolated BTK lesions, a successful endovascular procedure led to a high percentage of limb salvage at long-term follow-up.

Multiplex serum cytokine monitoring as a prognostic tool in rheumatoid arthritis.

OBJECTIVE: Early optimized therapy of rheumatoid arthritis (RA) results in improved outcomes. The initiation of optimized therapy is hindered by the difficulty of early diagnosis and the limitations of current disease activity and therapeutic response assessment tools. Identifying patients requiring early combination DMARD/biologic therapy is currently a significant clinical challenge given the lack of definitive prognostic criteria. Since cytokines are soluble intracellular signaling molecules that modulate disease pathology in RA, we tested the recent conjecture that en mass serum cyto-kine measurement and monitoring will provide a useful tool for effective therapeutic management in RA. METHODS: We assayed the levels of 16 serum cytokines in 18 RA patients treated prospectively with methotrexate and from 18 unaffected controls. Specific mechanistic aspects of inflammatory pathology in the periphery could be discerned on a patient-specific basis from patients' serum cytokine profiles, information that may aid in the design of anti-cytokine biologic therapy. A serum Cytokine Activity Index (CAI) was also created using multi-variant analysis methods. RESULTS: Distinct cytokines were significantly elevated in RA patients relative to controls, and three distinct clusters with correlations to disease activity were identified. The Cytokine Activity Index correlated well with the therapeutic res-ponse; responders and non-responders in this cohort were distinguishable as early as one month post initiation of methotrexate therapy, well before clinical assessments of response are commonly completed. CONCLUSION: Clinical assessment tools could be derived from this approach that may provide a means to continually track patients, allowing intervention strategies to be better evaluated on a patient-specific basis and to identify residual cytokine activity that could be used to guide combination therapy.

Circulating cytokines in Norwegian patients with psoriatic arthritis determined by a multiplex cytokine array system.

OBJECTIVES: Serum cytokines play an important role in the pathogenesis of psoriatic arthritis (PsA) by initiating and perpetuating various cellular and humoral autoimmune processes. The aim of this study was to describe a broad spectrum of T- and B-cell cytokines, growth factors and chemokines in patients with PsA and healthy individuals. METHODS: A novel protein array system, denoted as multiplex cytokine assay was utilized to measure simultaneously the levels of 23 circulating cytokines of patients with PsA and healthy individuals. Additionally, correlational clustering and discriminant function analysis (DFA), two multivariate, supervised analysis methods were employed to identify a subset of biomarkers in order to describe potential functional inter-relationships among these pathological cytokines and identify biomarkers with prognostic and diagnostic utility. RESULTS: Univariate analysis demonstrated that serum levels of a complex set of immune and inflammatory modulating cytokines are significantly up-regulated in patients with PsA relative to unaffected controls including interleukin (IL)-10, IL-13, interferen (IFN)-alpha, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor [CCL3 macrophage inflammatory protein (MIP)-1alpha], CCL4 (MIP-1beta) and CCL11 (Eotaxin), while granulocyte-colony stimulating factor was significantly reduced in PsA patients. Correlational clustering was able to discriminate among, and hence subclassify, patients with varying levels of disease activity, which may prove useful in guiding therapy in these apparently phenotypically distinct disease subsets. DFA identified EGF, IFN-alpha, VEGF, CCL3 (MIP-1alpha) and IL-12p40 as analytes with the strongest discriminatory power among various PsA patients and controls. CONCLUSIONS: Our findings suggest that these factors modulate PsA pathology and the articular involvement in a synergistic manner. Identifying factors could be used in the development of clinical diagnostic tests, which are valuable to guide evidence-based diagnosis and disease management of PsA.

Template-driven gene selection procedure.

The hierarchical clustering and statistical techniques usually used to analyse microarray data do not inherently represent the underlying biology. Herein, a hybrid approach involving characteristics of both supervised and unsupervised learning is presented. This approach is based on template matching in which the interaction of the variables of inherent malignancy and the ability to express the malignant phenotype are modelled. Immortalised normal urothelial cells and bladder cancer cells of different malignancy were grown in conventional two-dimensional tissue culture and in three dimensions on extracellular matrices (ECMs) that were either permissive or restrictive for expression of the malignant phenotype. The transcriptome represents the effects of two variables--inherent malignancy and the modulatory effect of ECM. By assigning values to each of the biological variables of inherent malignancy and the ability to express the malignant phenotype, a template was constructed, which encapsulated the interaction between them. Gene expression correlating both positively and negatively with the template was observed, but when iterative correlations were carried out, the different models for the template converged on the same actual template. A subset of 21 genes was identified, which correlated with two a priori models or an optimised model above the 95% confidence limits identified in a bootstrap resampling with 5000 permutations of the data set. The correlation coefficients of expression of several genes were > 0.8. Analysis of upstream transcriptional regulatory elements (TREs) confirmed that these genes were not a randomly selected set of genes. Several TREs were identified as significantly over-expressed in the sample of 20 genes for which TREs were identified, and the high correlations of several genes were consistent with transcriptional co-regulation. The authors suggest that the template method can be used to identify a unique set of genes for further investigation.

Genome-scale assessment of molecular pathology in systemic autoimmune diseases using microarray technology: a potential breakthrough diagnostic and individualized therapy-design tool.

Systemic autoimmune rheumatic diseases are of complex aetiology, characterized by an intricate interplay of various factors. A myriad of genes lies behind the heterogeneous manifestations of these diseases, and the overexpression and repression of particular genes form a specific gene-expression profile (genetic fingerprints) that is characteristic to the given disease phenotype. Besides the description of various cell types by using gene-expression profiling, the data should be directly applicable to the design of individual therapeutic protocols for patients suffering from various autoimmune diseases. In this review, we summarize the gene-expression profile, various genetic signatures of different autoimmune diseases and give an overview on the possible interpretations of the data. The application of recent breakthroughs in high-throughput molecular profiling technologies, such as microarray technology has been the basis for a revolution in biomedical research, as well as diagnostics and pharmaceutical development. It is easy to envision a day when personalized medicine, which is the diagnosis and treatment of a given patient with agents and procedures tailored to that patient's genetics, physiology and pathology, will become the standard of care.

A genome-scale assessment of peripheral blood B-cell molecular homeostasis in patients with rheumatoid arthritis.

OBJECTIVE: While rheumatoid arthritis (RA) is considered a prototypical autoimmune disease, the specific roles of B-cells in RA pathogenesis is not fully delineated. METHODS: We performed microarray expression profiling of peripheral blood B-cells from RA patients and controls. Data were analysed using differential gene expression analysis and 'gene networking' analysis (characterizing clusters of functionally inter-relelated genes) to identify both regulatory genes and the pathways in which they participate. Results were confirmed by quantitative real-time polymerase chain reaction and by measuring the levels of 10 serum cytokines involved in the pathways identified. RESULTS: Genes regulating and effecting the cell-cycle, proliferation, apoptosis, autoimmunity, cytokine networks, angiogenesis and neuro-immune regulation were differentially expressed in RA B-cells. Moreover, the serum levels of several soluble factors that modulate these pathways, including IL-1beta, IL-5, IL-6, IL-10, IL-12p40, IL-17 and VEGF were significantly increased in this cohort of RA patients. CONCLUSIONS: These results outline aspects of the multifaceted role B-cells play in RA pathogenesis in which immune dysregulation in RA modulates B-cell biology and thereby contributes to the induction and perpetuation of a pathogenic humoral immune response.

Transcriptional modulation of TCR, Notch and Wnt signaling pathways in SEB-anergized CD4+ T cells.

Gene expression changes in CD4 + Vbeta8+ T cells energized by in vivo exposure to staphylococcal enterotoxin B (SEB) bacterial superantigen compared to CD4 + Vbeta8+ non-energic T cells were assessed using DNA microarrays containing 5184 murine complementary DNAs. Anergy in splenic T cells of SEB-immunized BALB/c mice was verified by dramatically reduced proliferative capacity and an 8 x overexpression of GRAIL mRNA in CD4 + Vbeta8+ T cells taken from mice 7 days after injection. At an Associative t-test threshold of P<0.0005, 96 genes were overexpressed or detected only in anergic T cells, while 256 genes were suppressed or not detected in anergic T cells. Six of eight differential expressions tested using real-time quantitative PCR were validated. Message for B-Raf was detected only in non-anergic cells, while expression of the TCR signaling modulator Slap (Src-like adapter protein) and the TCR zeta-chain specific phosphatase Ptpn3 was enhanced. Modulation of multiple genes suggests downregulation of Wnt/beta-catenin signaling and enhanced Notch signaling in the anergic cells. Consistent with previous reports in a non-superantigen in vivo anergy model, mRNA for CD18 and the transcription factor Satb1 (special AT-rich-binding protein 1) was increased in SEB-energized T cells. This is the first report of global transcriptional changes in CD4+ T cells made anergic by superantigen exposure.

Mobile classification in microarray experiments.

In a homogeneous group of samples, there are genes whose expression variations can be attributed to factors other than experimental errors. These factors can include natural biological oscillations or metabolic processes. These genes are rarely classified as 'interesting' based on their variability profile. However, their dynamic behaviour can tease out important clues about naturally occurring biological processes in the organism under study and can be used for group classification. Dynamical discriminate function analysis was developed on the concept that stable classification parameters (roots) can be derived from highly variable gene-expression data. Stability of these combinations implies a strongly compensatory relationship that may divulge functional interconnections.

5alpha-Androstane-3alpha,17beta-diol activates pathway that resembles the epidermal growth factor responsive pathways in stimulating human prostate cancer LNCaP cell proliferation.

5alpha-Androstane-3alpha,17beta-diol (3alpha-diol) is considered to have no androgenic effects in androgen target organs unless it is oxidized to 5alpha-dihydrotestosterone (5alpha-DHT). We used microarray and bioinformatics to identify and compare 3alpha-diol and 5alpha-DHT responsive gene in human prostate LNCaP cells. Through a procedure called 'hypervariable determination', a similar set of 30 responsive genes involving signal transduction, transcription regulation, and cell proliferation were selected in 5alpha-DHT-, 3alpha-diol-, and epidermal growth factor (EGF)-treated samples. F-means cluster and networking procedures showed that the responsive pattern of these genes was more closely related between 3alpha-diol and EGF than between 5alpha-DHT and 3alpha-diol treatments. We conclude that 3alpha-diol is capable of stimulating prostate cell proliferation by eliciting EGF-like pathway in conjunction with androgen receptor pathway.

Apoptotic effect of rituximab on peripheral blood B cells in rheumatoid arthritis.

Rituximab (RTX) has proven efficacious in the treatment of rheumatoid arthritis (RA). Herein, we assessed the apoptosis-inducing capability of RTX in vitro on RA peripheral blood B-cell subsets and also compared the effects of RTX on B cells from rheumatoid factor-positive (RF+) and RF- patients. The likely relevance of B cells in disease was assessed by measuring B-cell-modulating serum cytokines. Peripheral blood B cells were isolated and cultured with the presence or absence of RTX. The levels of apoptosis within the naïve, memory and IgD+CD27+ B-cell subpopulations were determined by cytofluorometric analysis and caspase 3/7 assays. Levels of serum cytokines were measured with a multiplex cytokine array system. RTX induced significant apoptosis in all B-cell subsets in both RA and controls. In naïve and memory B cells from RA patients, RTX induced significantly higher levels of apoptosis than in controls. RTX induced apoptosis of B cells in RF+ and RF- patients. Serum levels of interleukin-1beta (IL-1beta), IL-4, IL-10 and IL-13 were profoundly increased in RF+ patients compared to RF- patients and controls. Although our cohort was small (10 RA patients), the data suggest that RTX induces apoptosis in all investigated subsets of B cells from RA patients. Interestingly, memory B cells from RA patients were more sensitive to RTX than memory cells from normal controls, suggesting that the delay in treatment response to RTX observed in clinical trials may be due in part to memory cell depletion. The apoptotic effects of RTX were similar in RF+ and RF- patients, but serum levels of B-cell-activating cytokine levels were only elevated in RF+ but not RF- patients. These data suggest that RTX is less effective in RF- RA because B cells play a less significant role in RA pathogenesis in RF- patients.

Circulating cytokines in primary Sjögren's syndrome determined by a multiplex cytokine array system.

Plasma cytokines play an important role in the pathogenesis of Sjögren's syndrome (SS) by initiating and perpetuating various cellular and humoural autoimmune processes. The aim of the present study was to describe a broad spectrum of T-cell and B-cell cytokines, growth factors, chemokines and molecules that could contribute to cell death in SS. A novel protein array system was utilized to measure simultaneously the levels of 25 plasma cytokines of patients with primary SS and healthy individuals. Furthermore, we correlated these plasma cytokine levels with potential laboratory and clinical parameters related to disease activity in SS. A subset of plasma cytokines [e.g. interleukin-1beta (IL-1beta), IL-6, CXCL8 (IL-8), IL-12 p40, IL-15, tumour necrosis factor-alpha (TNF-alpha), epidermal growth factor, CCL4 (MIP-1beta), CCL2 (MCP-1), CCL11 (Eotaxin), CCL5 (RANTES), TNF-RI and TNF-RII] was found to significantly differ between patients and controls. Also, distinct populations of cytokines were found to differentiate between patients with normal versus elevated ESR or IgG levels and patients with the presence or absence of extra-glandular manifestations (EGMs). Our results support the assumption that the multiplex cytokine array system can be successfully utilized in the diagnosis and disease management of SS. Furthermore, it may provide a powerful tool in the design of individualized anticytokine therapies.

Characterization of viable autofluorescent macrophages among cultured peripheral blood mononuclear cells.

BACKGROUND: Inflammatory macrophages that demonstrate intense autofluorescence have been isolated directly from alveolar and peritoneal tissues, but their generation in vitro remains vague. We use flow cytometry to identify a population of autofluorescent macrophages as they arise among nonadherent populations of cultured blood mononuclear cells. METHODS: Cells were obtained from donated blood buffy coats and placed in culture for 14 days. Unstained populations from the cells remaining in suspension were sampled daily using flow cytometry. During the first 5 culture days, a distinct population of autofluorescent cells arose and comprised an average of < or =14% of the total cell population. This population declined to less than 6% by culture day 8. RESULTS: The cells were identified as viable macrophages expressing CD68, lysozyme, and HLA-DR. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) demonstrated a unique cytokine profile with IL-1 alpha expression levels 138-fold higher than those measured in uncultured monocytes. No significant elevation in the levels of other cytokines was identified. Upon replating, the sorted populations became readherent, were able to ingest plastic beads, and remained viable for 6 or more additional weeks in culture without evidence of proliferation or multinucleation. CONCLUSION: Viable autofluorescent macrophage populations arising among cultured peripheral blood may be easily identified and isolated for further study using flow cytometry. Cytometry 44:38-44, 2001. Published 2001 Wiley-Liss, Inc.