CNS neurotrophins are biologically active and expressed by multiple cell types

Neutrotrophins are increasingly appreciated as potential modulators of neuronal function in the adult central nervous system (CNS). To describe the neurotrophin environment within the adult CNS, mRNA and protein expression patterns of neurotrophins-3 and –4 and of brain-derived neurotrophin were investigated in adult rat spinal cord and brain. Co-localization studies with CNS cell type-specific markers demonstrates that multiple cell types, including both neurons and glia, express these neurotrophins in the normal adult CNS. Although widely implicated in important CNS functions such as synaptic plasticity, biological activity of endogenous CNS neurotrophins has not been directly demonstrated. With a sensitive neurite outgrowth bioassay we demonstrate that CNS neurotrophins elicit neurite outgrowth and are biologically active. Moreover, antibody-blocking studies suggest that these three neurotrophins may comprise the bulk of adult CNS neurotrophic activity.     

Chemokines derived from soluble fusion proteins expressed in Escherichia coli are biologically active

Chemokines are a class of low molecular weight proteins that are involved in leukocytes trafficking. Due to their involvement in recruiting immune cells to sites of inflammation, chemokines, and chemokine receptors have become an attractive class of therapeutic targets. However, when expressed in Escherichia coli chemokines are poorly soluble and accumulate in inclusion bodies. Several purification methods have been described but involve time-consuming refolding, buffer exchange, and purification steps that complicate expression of these proteins. Here, we describe a simple and reliable method to express chemokines as fusions to the protein NusA. The fusion proteins were largely found in the soluble fraction and could be readily purified in a single step. Proteolytic cleavage was used to obtain soluble recombinant chemokines that were found to be very active in a novel in vitro chemotaxis assays. This method could be applied to several alpha and beta human chemokines, suggesting that it is generally applicable to this class of proteins.     

Clinical value of whole-body PET/CT in patients with active rheumatic diseases

Advanced imaging techniques may enable early diagnosis and monitoring of therapy in various rheumatic diseases. To prevent irreversible tissue damage, inflammatory rheumatic disease must be diagnosed and treated in pre-clinical stages, requiring highly sensitive detection techniques. Positron emission tomography (PET) provides highly sensitive, quantitative imaging at a molecular level, revealing the important pathophysiological processes underlying inflammation. This review provides an overview of the current utility of ¹⁸?F-fluorodeoxyglucose (FDG)-PET/computed tomography (CT) in patients with active rheumatic diseases such as rheumatoid arthritis, spondyloarthritis, polymyalgia rheumatica, adult-onset Still’s disease, relapsing polychondritis, immunoglobulin G4-related disease, large-vessel vasculitis, Wegener’s granulomatosis, polymyositis, and dermatomyositis. We also discuss the role of FDG-PET/CT in the diagnosis and monitoring of these diseases.     

Clinical value of whole-body PET/CT in patients with active rheumatic diseases

Advanced imaging techniques may enable early diagnosis and monitoring of therapy in various rheumatic diseases. To prevent irreversible tissue damage, inflammatory rheumatic disease must be diagnosed and treated in pre-clinical stages, requiring highly sensitive detection techniques. Positron emission tomography (PET) provides highly sensitive, quantitative imaging at a molecular level, revealing the important pathophysiological processes underlying inflammation. This review provides an overview of the current utility of ¹⁸ F-fluorodeoxyglucose (FDG)-PET/computed tomography (CT) in patients with active rheumatic diseases such as rheumatoid arthritis, spondyloarthritis, polymyalgia rheumatica, adult-onset Still’s disease, relapsing polychondritis, immunoglobulin G4-related disease, large-vessel vasculitis, Wegener’s granulomatosis, polymyositis, and dermatomyositis. We also discuss the role of FDG-PET/CT in the diagnosis and monitoring of these diseases.     

Renal co-morbidity in patients with rheumatic diseases

Renal co-morbidity is common in patients with rheumatic disease based on regular assessment of serum and urine parameters of renal function. When patients present with both arthritis and renal abnormalities the following questions have to be addressed. Is kidney disease a complication of rheumatic disease or its management, or are they both manifestations of a single systemic autoimmune disease? Is rheumatic disease a complication of kidney disease and its management? How do rheumatic disease and kidney disease affect each other even when they are unrelated? The present review provides an overview of how to address these questions in daily practice.     

Renal co-morbidity in patients with rheumatic diseases

Renal co-morbidity is common in patients with rheumatic disease based on regular assessment of serum and urine parameters of renal function. When patients present with both arthritis and renal abnormalities the following questions have to be addressed. Is kidney disease a complication of rheumatic disease or its management, or are they both manifestations of a single systemic autoimmune disease? Is rheumatic disease a complication of kidney disease and its management? How do rheumatic disease and kidney disease affect each other even when they are unrelated? The present review provides an overview of how to address these questions in daily practice.     

Cardiovascular co-morbidity in patients with rheumatic diseases

During recent years atherosclerosis, the major cause of cardiovascular disease (CVD), has been recognised as a chronic inflammatory condition in which rupture of atherosclerotic lesions appears to play a major role. The risk of CVD is raised in many rheumatic diseases. This risk is high in systemic lupus erythematosus - as much as a 50-times increase among middle-aged women has been reported. Studies on CVD and atherosclerosis in rheumatic disease could thus provide interesting information about CVD and atherosclerosis in addition to being an important clinical problem. A combination of traditional and nontraditional risk factors accounts for the increased risk of CVD and atherosclerosis in rheumatic disease. One interesting possibility is that atherosclerotic lesions in rheumatic disease are more prone to rupture than normal atherosclerotic lesions. It is also likely that increased risk of thrombosis may play an important role, not least in systemic lupus erythematosus. Further, it is not clear whether an increased risk of CVD is a general feature of rheumatic disease, or whether this only occurs among subgroups of patients. It should be emphasised that there is an apparent lack of treatment studies where CVD in rheumatic disease is the end point. Control of disease activity and of traditional risk factors, however, appears to be well founded in relation to CVD in rheumatic disease. Further studies are needed to determine the exact role of lipid-lowering drugs as statins. Hopefully novel therapies can be developed that target the causes of the inflammation in atherosclerotic lesions both in rheumatic patients and in the general population.     

Androgen binding protein is tissue-specifically expressed and biologically active in transgenic mice

In view of the inconclusive data concerning the role of androgen-binding protein (ABP) in male reproductive physiology, we thought it would be pertinent to make several transgenic mouse lines overexpressing the rat ABP gene to unravel its role in Sertoli cell and epididymal homeostasis. Heterozygote transgenic mouse lines carrying the 5.5 kb ABP rat genomic DNA were produced by pronuclear microinjection. Northern blot analysis showed overexpression of rat ABP (rABP) mRNA in the testis of transgenic mice compared to rat testis control. rABP was appropriately expressed in Sertoli cells as demonstrated by in situ hybridization analysis. Sertoli cell number is increased in the seminiferous tubules of mice overexpressing rABP compared to non-transgenic littermates and scattered Sertoli cells present vacuolated-like cytoplasms, PAS and osmium negative. Compared to the wild type, the transgenic mice exhibited reduced fertility and focal damage in seminiferous epithelium characterized by morphological features compatible with programmed cell death.     

Angiogenesis and chemokines in rheumatoid arthritis and other systemic inflammatory rheumatic diseases

Angiogenesis, the formation of new vessels, is important in the pathogenesis of rheumatoid arthritis (RA) and other inflammatory diseases. Chemotactic cytokines termed chemokines mediate the ingress of leukocytes, including neutrophils and monocytes into the inflamed synovium. In this review, authors discuss the role of the most important angiogenic factors and angiogenesis inhibitors, as well as relevant chemokines and chemokine receptors involved in chronic inflammatory rheumatic diseases. RA was chosen as a prototype to discuss these issues, as the majority of studies on the role of angiogenesis and chemokines in inflammatory diseases were carried out in arthritis. However, other systemic inflammatory (autoimmune) diseases including systemic lupus erythematosus (SLE), systemic sclerosis (SSc), Sjögren's syndrome (SS), mixed connective tissue disease (MCTD), polymyositis/dermatomyositis (PM/DM) and systemic vasculites are also discussed in this context. As a number of chemokines may also play a role in neovascularizaton, this issue is also described here. Apart from discussing the pathogenic role of angiogenesis and chemokines, authors also review the regulation of angiogenesis and chemokine production by other inflammatory meditors, as well as the important relevance of neovascularization and chemokines for antirheumatic intervention.     

Recombinant human fibrinogen expressed in the yeast Pichia pastoris was assembled and biologically active

Fibrinogen is a large plasma glycoprotein with a molecular mass of 340 kDa that plays a critical role in the final stage of blood coagulation. Human plasma fibrinogen is a dimeric molecule comprising two sets of three different polypeptides (Aα, 66 kDa; Bβ, 55 kDa; γ, 48 kDa). To express recombinant human fibrinogen in the methylotrophic yeast Pichia pastoris, we constructed an expression vector containing three individual fibrinogen chain cDNAs under the control of the mutated AOX2 (mAOX2) promoter. First, P. pastoris GTS115 was transformed with the vector, but the expressed recombinant fibrinogen suffered severe degradation by yeast-derived proteases under conventional nutrient culture conditions. Fibrinogen degradation was prevented by using the protease A-deficient strain SMD1168 as a host strain and regulating the pH of the culture to between 5.5 and 7.0. Western blot analysis revealed that the Aα, Bβ and γ chains of recombinant fibrinogen were assembled and secreted as a complete molecule. The Bβ chain of the recombinant fibrinogen was N-glycosylated but the Aα chain, as in plasma fibrinogen, was not. The γ chains however were heterologous, one being N-glycosylated and the other not. The recombinant fibrinogen was capable of forming a thrombin-induced clot in the presence of factor XIIIa and both the glycosylated and the non-glycosylated γ chains were involved in the formation of cross-linking fibrin. The present study indicates that the recombinant fibrinogen expressed in P. pastoris, although different from plasma fibrinogen in post-translational modification, is correctly assembled and biologically active.     

Isolation of specific and biologically active peptides that bind cells from patients with acute myeloid leukemia (AML)

Imatinib was the first BCR-ABL-targeted agent approved for the treatment of patients with chronic myeloid leukemia (CML) and confers significant benefit for most patients; however, a substantial number of patients are either initially refractory or develop resistance. Point mutations within the ABL kinase domain of the BCR-ABL fusion protein are a major underlying cause of resistance. Of the known imatinib-resistant mutations, the most frequently occurring involve the ATP-binding loop (P-loop). In vitro evidence has suggested that these mutations are more oncogenic with respect to other mutations and wild type BCR-ABL. Dasatinib and nilotinib have been approved for second-line treatment of patients with CML who demonstrate resistance (or intolerance) to imatinib. Both agents have marked activity in patients resistant to imatinib; however, they have differential activity against certain mutations, including those of the P-loop. Data from clinical trials suggest that dasatinib may be more effective vs. nilotinib for treating patients harboring P-loop mutations. Other mutations that are differentially sensitive to the second-line tyrosine kinase inhibitors (TKIs) include F317L and F359I/V, which are more sensitive to nilotinib and dasatinib, respectively. P-loop status in patients with CML and the potency of TKIs against P-loop mutations are key determinants for prognosis and response to treatment. This communication reviews the clinical importance of P-loop mutations and the efficacy of the currently available TKIs against them.     

Marine Pseudoalteromonas species are associated with higher organisms and produce biologically active extracellular agents

The newly established genus Pseudoalteromonas contains numerous marine species which synthesize biologically active molecules. The production of a range of compounds which are active against a variety of target organisms appears to be a unique characteristic for this genus and may greatly benefit Pseudoalteromonas cells in their competition for nutrients and colonization of surfaces. Species of Pseudoalteromonas are generally found in association with marine eukaryotes and display anti-bacterial, bacteriolytic, agarolytic and algicidal activities. Moreover, several Pseudoalteromonas isolates specifically prevent the settlement of common fouling organisms. While a wide range of inhibitory extracellular agents are produced, compounds promoting the survival of other marine organisms living in the vicinity of Pseudoalteromonas species have also been found.     

Plasma levels of BAFF and APRIL are elevated in patients with asthma in Saudi Arabia

B-cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) are members of the tumor necrosis factor superfamily of cytokines and can induce B cell activation, differentiation, and antibody production via interaction with their receptors, including transmembrane activator, calcium modulator, and cyclophilin ligand interactor (TACI), B-cell maturation antigen (BCMA), and B-cell activating factor receptor (BAFF-R). Herein, we assessed the plasma protein levels of BAFF and APRIL in patients with asthma to determine whether their expression is correlated with total IgE production and examined the surface expression of BAFF/APRIL receptors on B cells. Blood samples were collected from 47 patients with controlled asthma symptoms and 20 healthy normal controls, and plasma levels of APRIL, BAFF, and total IgE protein were quantified by corresponding ELISA assays. Furthermore, lymphocytes were isolated and B cells were analyzed for the presence of BAFF-R, BCMA, and TACI receptors using flow cytometry. Our results showed that IgE, BAFF, and APRIL plasma levels were markedly increased in patients with asthma compared with healthy controls. Moreover, expression of BAFF-R and BCMA, but not that of TACI, was significantly increased in patients with asthma compared with healthy controls. Overall, the findings suggest BAFF and APRIL as key mediators of asthma, and determination of their plasma levels may be useful in monitoring asthma symptoms and treatment response.     

Analogs of pancreatic polypeptide and peptide YY with a locked PP-fold structure are biologically active

Pancreatic polypeptide (PP), peptide YY (PYY) and neuropeptide Y (NPY), members of the PP-fold family share a high degree of sequence homology. Nuclear magnetic resonance (NMR) and X-ray crystallography studies have shown these peptides can adopt a tightly organized tertiary structure called the PP-fold, which has long been assumed to be the active structure of this family of peptides. To date, however, no studies have been completed with PYY and PP which confirm if the PP-fold structure is important for their physiological actions. The aim of the study was to test if PYY and PP locked into the PP-fold maintained biological activity. Therefore, we designed and produced analogs of PP and PYY in a cyclic conformation with two cysteine amino acid substitutions at the N-terminus and at position 27. These were oxidized to form a cysteine disulfide bond locking the peptides into the PP-fold structure. Studies demonstrate that the cyclic analogs have both similar in vivo activity to their parent molecules, and affinity for the Y2 and Y4 receptors. Results suggest that the proposed PP and PYY-fold is likely to be their biologically active conformation.     

Differential effects on BAFF and APRIL levels in rituximab-treated patients with systemic lupus erythematosus and rheumatoid arthritis

The objective of this study was to investigate the interaction between levels of BAFF (B-cell activation factor of the tumour necrosis factor [TNF] family) and APRIL (a proliferation-inducing ligand) and B-cell frequencies in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) treated with the B-cell-depleting agent rituximab. Ten patients with SLE were treated with rituximab in combination with cyclophosphamide and corticosteroids. They were followed longitudinally up to 6 months after B-cell repopulation. Nine patients with RA, resistant or intolerant to anti-TNF therapy, treated with rituximab plus methotrexate were investigated up to 6 months after treatment. The B-cell frequency was determined by flow cytometry, and serum levels of BAFF and APRIL were measured by enzyme-linked immunosorbent assays. BAFF levels rose significantly during B-cell depletion in both patient groups, and in patients with SLE the BAFF levels declined close to pre-treatment levels upon B-cell repopulation. Patients with SLE had normal levels of APRIL at baseline, and during depletion there was a significant decrease. In contrast, patients with RA had APRIL levels 10-fold higher than normal, which did not change during depletion. At baseline, correlations between levels of B cells and APRIL, and DAS28 (disease activity score using 28 joint counts) and BAFF were observed in patients with RA. In summary, increased BAFF levels were observed during absence of circulating B cells in our SLE and RA patient cohorts. In spite of the limited number of patients, our data suggest that BAFF and APRIL are differentially regulated in different autoimmune diseases and, in addition, differently affected by rituximab treatment.     

Vasculopathy and disordered angiogenesis in selected rheumatic diseases: rheumatoid arthritis and systemic sclerosis

Angiogenesis is important in the pathogenesis of systemic inflammatory rheumatic diseases, a family of related disorders that includes rheumatoid arthritis and systemic sclerosis. Rheumatoid arthritis is the rheumatic disease in which the role of angiogenesis has been studied most extensively. However, whereas rheumatoid arthritis is characterized by excessive angiogenesis, the situation is not as clear cut in other rheumatic diseases. For example, systemic sclerosis is characterized by reduced capillary density with insufficient angiogenic responses. Results with angiogenesis inhibitors are controversial, and there is--in parallel--a wide range of upregulated angiogenic factors such as vascular endothelial growth factor. Dysregulation of angiogenesis in systemic sclerosis is accompanied by other pathogenic processes, including fibrosis, autoimmunity and vasculopathy. Animal models with at least partial features of the vasculopathy observed in systemic sclerosis include wound healing models, graft versus host disease models and, in particular, the University of California at Davis line 200 chicken model of systemic sclerosis.     

Production of biologically active recombinant goose FSH in a single chain form with a CTP linker sequence

FSH is a glycoprotein hormone secreted by the pituitary gland that is essential for gonadal development and reproductive function. In avian reproduction study, especially in avian reproduction hormone study, it is hindered by the lack of biologically active FSH. In order to overcome this shortcoming, we prepared recombinant goose FSH as a single chain molecule and tested its biological activities in the present study. Coding sequences for mature peptides of goose FSH α and β subunits were amplified from goose pituitary cDNA. A chimeric gene containing α and β subunit sequences linked by the hCG carboxyl terminal peptide coding sequence was constructed. The recombinant gene was inserted into the pcDNA3.1-Fc eukaryotic expression vector to form pcDNA-Fc-gFSHβ-CTP-α and then transfected into 293-F cells. A recombinant, single chain goose FSH was expressed and verified by SDS-PAGE and western blot analysis, and was purified using Protein A agarose affinity and gel filtration chromatography. Biological activity analysis results showed that the recombinant, chimeric goose FSH possesses the function of stimulating estradiol secretion and cell proliferation, in cultured chicken granulosa cells. These results indicated that bioactive, recombinant goose FSH has been successfully prepared in vitro. The recombinant goose FSH will have the potential of being used as a research tool for studying avian reproductive activities, and as a standard for developing avian FSH bioassays.     

Interleukin-2 is present in human blood vessels and released in biologically active form by heparanase

Interleukin-2 (IL-2) is a multifaceted cytokine with immunostimulatory and immunosuppressive properties. Our laboratory recently demonstrated that the availability of IL-2 is regulated, in part, by association with perlecan, a heparan sulfate proteoglycan. Given the abundance of perlecan in blood vessels, we asked whether IL-2 is present in vessel walls. Our results indicate that IL-2 is associated with endothelial and smooth muscle cells within the human arterial wall. This IL-2 is released by heparanase, and promotes the proliferation of an IL-2-dependent cell line. Given the presence of IL-2 in human arteries, we asked whether the large vessels of IL-2-deficient mice were normal. The aortas of IL-2-deficient mice exhibited a loss of smooth muscle cells, suggesting that IL-2 may contribute to their survival. In their entirety, these results suggest a here-to-fore unrecognized role of IL-2 in vascular biology, and have significant implications for both the immune and cardiovascular systems.     

Contents of antibodies to Bordetella pertussis antigens in patients with rheumatic diseases

Study of presence of antibodies against pertussis in 72 rheumatic patients (with uvenile rheumatoid arthritis, systemic lupus erythematosus, etc.) aged 1-18 year old without history of pertussis was performed. Mean age of the patients was 10.6 +/- 0.48 year old, duration of illness--51.2 +/- 4.42 months. Immunosupressive therapy at the time of the study was conducted in 68 (94.4%) children. Using ELISA method, IgG to pertussis toxin (PT) and to antigens of acellular pertussis vaccine (aPV) were detected in 98.6% and 100% of children. High titers of antibodies were detected more frequently in 7-18 year old age group, which can indicate recent pertussis disease or infection. Vaccination history was studied in 131 children with rheumatic diseases. Incidence of pertussis in 43 unvaccinated children was 116.3 per 1000, and in 16 children with incomplete vaccination--62.5 per 1000. Out of 75 patients, who received vaccination series and revaccination, clinically distinct pertussis was not diagnosed.