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Impaired immunogenicity of COVID-19 vaccinations in inflammatory arthritis (IA) patients results in diminished immunity. However, optimal booster vaccination regimens are still unknown. Therefore, this study aimed to assess the kinetics of humoral and cellular responses in IA patients after the COVID-19 booster. In 29 IA patients and 16 healthy controls (HC), humoral responses (level of IgG antibodies) and cellular responses (IFN-γ production) were assessed before (T0), after 4 weeks (T1), and after more than 6 months (T2) from the booster vaccination with BNT162b2. IA patients, but not HC, showed lower anti-S-IgG concentration and IGRA fold change at T2 compared to T1 ( p = 0.026 and p = 0.031). Furthermore, in IA patients the level of cellular response at T2 returned to the pre-booster level (T0). All immunomodulatory drugs, except IL-6 and IL-17 inhibitors for the humoral and IL-17 inhibitors for the cellular response, impaired the immunogenicity of the booster dose at T2. Our study showed impaired kinetics of both humoral and cellular responses after the booster dose of the COVID-19 vaccine in IA patients, which, in the case of cellular response, did not allow the vaccination effect to be maintained for more than 6 months. Repetitive vaccination with subsequent booster doses seems to be necessary for IA patients.

Introduction: Previous studies have shown a reduction in the effectiveness of primary COVID-19 vaccination in patients with rheumatic diseases. However, limited data is available regarding the effectiveness of the COVID-19 vaccine booster dose, especially on cellular response. The study aimed to assess the humoral and cellular immunogenicity of a booster dose in patients with inflammatory arthritis (IA)., Patients and Methods: 49 IA and 47 age and sex-matched healthy controls (HC) were included in a prospective cohort study. Both groups completed primary COVID-19 vaccination and after more than 180 days received a BNT162b2 booster shot. Humoral responses (level of IgG antibodies) and cellular responses (IFN-γ production) were assessed before and after 4 weeks from the booster dose of the vaccine., Results: After the booster dose, all participants showed an increased humoral response, although significantly reduced antibody levels were observed in IA patients compared to HC (p=0.004). The cellular response was significantly lower both before (p<0.001) and after (p<0.001) the booster dose in IA patients as compared to HC. Among the immunomodulatory drugs, only biological and targeted synthetic drugs lowered the humoral response after booster vaccination. However, the cellular response was decreased after all immunomodulatory drugs except IL-17 inhibitors and sulfasalazine., Conclusion: Our data indicate that patients with rheumatic diseases present lower humoral and cellular responses after the COVID-19 booster vaccine in comparison to HC. This may translate into a recommendation for subsequent booster doses of the COVID-19 vaccine for rheumatic patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wroński, Jaszczyk, Roszkowski, Felis-Giemza, Bonek, Kornatka, Plebańczyk, Burakowski, Lisowska, Kwiatkowska, Maśliński, Wisłowska, Massalska, Ciechomska and Kuca-Warnawin.)

This study aimed to investigate the associations of microRNA (miRs) signatures with cytokines, serum lipids, and disease activity in patients with psoriatic arthritis (PsA), ankylosing spondylitis (AS), and rheumatoid arthritis (RA). In total, 65 patients (PsA n = 25, AS n = 25, RA n = 15) and 25 healthy controls (HC) were enrolled into the study. The expression of miR-223-5p, miR-92b-3p, miR-485-3p, miR-10b-5p, let-7d-5p, miR-26a-2-3p, miR-146b-3p, and cytokines levels were measured in sera. DIANA-mirPath analysis was used to predict pathways targeted by the dysregulated miRs. Disease activity scores were calculated. Lipid profile, uric acid, glucose level, and C-reactive protein (CRP) concentrations were determined in the blood. Based on lipid profiles, the PsA group had hypertriglyceridaemia, and RA patients revealed mixed dyslipidaemia, while in AS, no specific changes were found. miR expression analysis revealed upregulation of miR-26a-2-3p and miR-10b-5p in PsA, miR-485-3p in AS, and let-7d-5p in RA. Several correlations between disease activity indexes, metabolites levels, and expression of miRs were observed in PsA, RA, and AS patients. Finally, in ROC analysis, miR-26a-2-3p/miR-485-3p, and let-7d-5p/miR-146b-3p tandems revealed high sensitivity and specificity in distinguishing between PsA, AS, and RA. Our study illustrates the superiority of miR expressions in distinguishing between RA, PsA, and AS. In PsA, a unique regulatory pathway exists through miR-26a-2-3p, miR-223-5p, miR-10b-5p, and miR-92b-3p that converges proatherogenic metabolism and disease activity.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are characterized by joint and systemic high- or low-grade inflammation, respectively. Adipose tissue (AT) may contribute to the pathogenesis of these diseases. To address this issue, we investigated whether basal and pro-inflammatory cytokine (IL-1β)-triggered release of adipocytokines (TNF, IL-6, IL-10, IL-1Ra, TGFβ, CCL2/MCP-1, CCL5/RANTES, MMP-3) from subcutaneous (ScAT) and intraarticular (AAT) adipose tissues of RA and OA patients mirror differences between these diseases in an intensity of systemic and local inflammation. We found that in both diseases basal adipocytokine release was usually higher from AAT than ScAT, reflecting stronger local than systemic inflammation. However, ScAT secreted considerable amounts of pro- and anti-inflammatory factors as well. Spontaneous secretion of some adipocytokines (MMP-3 and/or TNF, CCL2/MCP-1, IL-1Ra) was higher in osteoarthritis than rheumatoid ATs and probably caused by weaker anti-inflammatory treatment of OA patients. By contrast, reactivity of ATs to IL-1β was significantly lower in OA than RA and IL-1β antagonist (IL-1Ra) could be responsible for this because we found its overproduction in OA ATs. Interestingly, higher reactivity of ScAT than AAT to IL-1β was a characteristic for OA while reactivity of rheumatoid ScAT and AAT to this stimulus was equal. We conclude that differences between OA and RA in reactivity of AAT and ScAT to pro-inflammatory stimulus mimicking in vivo condition reflect dissimilarity in an intensity of disease-specific inflammation and thus support contribution of ATs to these pathological processes. Moreover, we propose that more efficient anti-inflammatory mechanism(s) are preserved in ATs of OA than RA patients.

Objectives: Enhanced/disturbed activities of monocytes are crucial for perpetuation and for development of rheumatoid arthritis (RA). Therefore, knowledge about monocyte activities and regulation of molecular pathways operating within monocytes early in the course of RA development may help to predict the progression to the full-blown disease. We aimed to investigate the profile of miRNAs expression in circulating monocytes and monocyte-related cytokines in sera of individuals at undifferentiated arthritis (UA) stage, wich could serve as new biomarkers for RA development., Material and Methods: Magnetically sorted monocytes from peripheral blood of 20 UA patients served for total RNA isolation. RNA samples were used for microRNA profiling. Concentrations of CCL3/MIP-1α, M-CSF, CCL2/MCP-1, IL-6, TNF-α and IL-15 in sera of UA patients were measured using ELISA assays. Verification of diagnosis after 4 years of follow-up led to the identification of patients who developed RA (UA→RA patients) and patients who remained still in UA phase (UA → UA patients). Comparisons between patients groups were performed using two-tailed Mann-Whitney U test., Results: We identified 50 miRNAs in monocytes with the largest variation of expression across all patients samples. From these selected miRNAs, expression of miR-642b-5p, miR-483-3p, miR-371b-5p were significantly up-regulated and miR-25-3p and miR-378d were significantly down-regulated in UA → RA vs. UA → UA patients. This specific pattern of miRNAs expression in circulating monocytes paralleled elevated IL-15 and M-CSF concentrations in sera of UA patients who progressed to RA., Conclusions: Results of our pilot study indicate that altered activity of monocytes can be detected at early stages of RA. We found new miRNA candidates differentially expressed in peripheral blood monocytes and elevated concentrations of IL-15 and M-CSF involved in monocyte activity and differentiation in patients with UA who subsequently developed RA, in comparison to UA patients who did not progress to RA after 4 years follow-up., Competing Interests: The authors declare no conflict of interest.

Rheumatoid arthritis (RA), which is a chronic inflammatory disease with a multifactorial aetiology, leads to partial or permanent disability in the majority of patients. It is characterised by persistent synovitis and formation of pannus, i.e. invasive synovial tissue, which ultimately leads to destruction of the cartilage, subchondral bone, and soft tissues of the affected joint. Moreover, inflammatory infiltrates in the subchondral bone, which can lead to inflammatory cysts and later erosions, play an important role in the pathogenesis of RA. These inflammatory infiltrates can be seen in magnetic resonance imaging (MRI) as bone marrow oedema (BME). BME is observed in 68-75% of patients in early stages of RA and is considered a precursor of rapid disease progression. The clinical significance of synovitis and bone marrow oedema as precursors of erosions is well established in daily practice, and synovitis, BME, cysts, hyaline cartilage defects and bone erosions can be detected by ultrasonography (US) and MRI. A less explored subject is the inflammatory and destructive potential of intra- and extra-articular fat tissue, which can also be evaluated in US and MRI. Finally, according to certain hypotheses, hyaline cartilage damage may trigger synovitis and lead to irreversible joint damage, and MRI may be used for preclinical detection of cartilage biochemical abnormalities. This review discusses the pathomechanisms that lead to articular cartilage and bone damage in RA, including erosion precursors such as synovitis and osteitis and panniculitis, as well as the role of imaging techniques employed to detect early cartilage damage and bone erosions., Competing Interests: The authors declare no conflict of interest.

Background: Rheumatoid arthritis (RA) is a systemic, autoimmune disease leading to joint destruction and ultimately disability. Bone marrow (BM) is an important compartment in RA, where pathological processes from "outside the joint" can occur. IL-17 is a cytokine that exerts proinflammatory effects and participates in the process of bone destruction. It is believed that IL-17 is involved in pathogenesis of RA. However, little is known about the biology of this cytokine in BM. In the present study we investigated Th17-related cytokines in RA BM., Methods: BM samples were obtained from RA and osteoarthritis (OA) patients during total hip replacement surgery. Levels of IL-17AF, IL-17AA, IL-17FF, IL-1β, IL-6, IL-23, TGF-β and CCL20 in BM plasma were determined by specific enzyme-linked immunosorbent assay tests. Percentage of IL-17-producing cells in BM was evaluated by flow cytometry. The effect of IL-15 stimulation on IL-17 production by BM mononuclear cells was examined in vitro., Results: Increased levels of IL-17AF were observed in BM plasma of RA patients in comparison to OA patients. Increased concentrations of IL-1β, IL-6 and CCL20 were observed in RA compared to OA BM plasma. Concordant with these findings, significantly increased percentages of CD3 + CD4 + IL-17 + and CD3 + CD4 + IL-17 + IFN-γ + cells were present in RA BM in comparison to OA BM samples. Finally, abundant in RA BM, IL-15 increased IL-17 production by cultured BM mononuclear cells., Conclusions: In the course of RA, the BM microenvironment can promote the development of Th17 cell responses and overproduction of IL-17AF that may lead to increased inflammation and tissue destruction in RA BM.

In the general population, low-grade inflammation of adipose tissue accompanies obesity and contributes to cardiovascular disease (CVD) development, but the implication of this tissue in rheumatic disease pathology is unclear. Therefore, we characterized the secretory activity of subcutaneous abdominal adipose tissue (SAAT) of females with rheumatoid arthritis (RA) and osteoarthritis (OA) and searched for its relationship with intensity of systemic inflammation, body composition and comorbidity. The secretion of classical adipokines (leptin, adiponectin), pro- and anti-inflammatory factors, i.e. interleukin (IL)-6, IL-8, IL-10, tumour necrosis factor (TNF), macrophage migration inhibitory factor (MIF) and hepatocyte growth factor (HGF), from SAAT explants was measured by specific enzyme-linked immunosorbent assays. Patients' body composition was evaluated by bioelectric impendence technique. Rheumatoid SAAT secreted more adiponectin, IL-6, IL-10, TNF and MIF but less leptin than respective osteoarthritis tissues. In RA patients, TNF secretion correlated with cachectic body composition, HGF release was linked to secondary amyloidosis and visceral fat rating was an independent risk factor for CVD. In OA, secretion of leptin and HGF positively, while adiponectin inversely, correlated with systemic inflammation markers, and the release of MIF was an independent risk factor for CVD. This study reveals differences between RA and OA patients in SAAT secretory activity and suggests its different clinical impact in these diseases, characterized by high- and low-grade systemic inflammation, respectively. In RA, SAAT may directly or via an effect on body composition contribute to amyloidosis, cachexia or CVD co-occurring, while in OA SAAT-derived adipocytokines may rather regulate intensity of systemic inflammation and redound to CVD emergence.

The most specific autoimmunity known for rheumatoid arthritis (RA) is reflected by generation of anti-citrullinated protein antibodies (ACPA). Presence of ACPA in established RA is associated with disease severity, while generation of ACPA at early developmental phases of RA can have a strong predictive value for progressing to the full-blown disease. Hence, development of ACPA may be of crucial importance to the pathogenesis of RA. Therefore, a lot of effort has been put recently to investigate the feature of ACPA at early developmental stages of RA (before disease onset) and functional activities of these autoantibodies. Results of these studies enlarged the knowledge about the nature of ACPA, which is essential for planning the therapeutic or preventive strategies interfering with their development and pathogenic functions. In this review we describe recent evidence for a role of ACPA in the etiopathogenesis of RA and indicate key unresolved issues regarding ACPA biology that need to be clarified in the future., Competing Interests: The authors declare no conflict of interest.

Objectives: Inflammatory bowel disease (IBD) and spondyloarthritis (SpA) have some overlapping clinical features, i.e. gut and joint inflammation. Cytokines of interleukin 17(IL-17)/IL-23 axis play a pathogenic role in both diseases. Integrins (ITGs) regulate migration of immune cells to inflamed tissues (ITGβ7 into gut, ITGβ2 into gut and also to other tissues). In this study, we search for differences in the serum concentrations of these cytokines and integrins between patients suffering from SpA or IBD with and without overlapping symptoms., Material and Methods: Patients with SpA ( n = 30), IBD ( n = 68), and healthy volunteers ( n = 28) were included in the study. Fourteen SpA patients reported symptoms characteristic for IBD. Spondyloarthritis symptoms were diagnosed in 50% of IBD patients, while other patients of this group reported arthralgia only. Serum concentrations of IL-17, IL-22, IL-23, ITGβ2, and ITGβ7 were measured by specific enzyme-linked immunosorbent assay using commercially available sets. The Mann-Whitney and Spearman's rank tests were used for intergroup comparison and correlation assessment, respectively., Results: Comparison of patient groups showed significantly higher serum concentrations of IL-17, IL-22, and ITGβ7 in SpA, and up-regulated levels of IL-23 in IBD patients. Similar differences were observed between patient subgroups, both with and without overlapping symptoms. In SpA but not in IBD patients, serum concentrations of ITGβ7 inversely correlated ( r = -0.552) with C-reactive protein., Conclusions: Patients with SpA and IBD differ in the circulating concentrations of IL-17/IL-23 axis cytokines and ITGβ7, irrespectively of the presence or absence of overlapping symptoms. Therefore, we conclude that observed differences are attributed rather to underlying than concurrent disease., Competing Interests: The authors declare no conflict of interest.

Objectives: Rheumatoid arthritis (RA) is a chronic inflammatory disease leading to joint destruction. In addition to involvement of the joints, there is growing evidence that inflammatory/autoimmune processes take place in bone marrow, beginning the disease onset. Activated T and B cells accumulate in bone marrow, where also effective antigen presentation takes place. An increased number of activated T cells was observed in RA in comparison to osteoarthritis (OA) bone marrow. In the present study we analyzed the levels of chemokines that may be responsible for accumulation/retention of T-cells in the bone marrow of RA and OA patients., Material and Methods: Bone marrow samples were obtained from RA and OA patients during total hip replacement surgery, and bone marrow plasma was obtained by gradient centrifugation. Levels of the chemokines CX3CL1, CCL5, CCL2, CXCL12 and CXCL1 were measured in bone marrow plasma by specific ELISAs. Comparison between the groups of patients and statistical significance were analyzed by the two-tailed Mann-Whitney U test., Results: Increased levels of CX3CL1 (818 ±431 pg/ml vs. 502 ±131 pg/ml, p < 0.0007) and CCL5 (5967 ±1680 pg/ml vs. 4878 ±2360 pg/ml, p < 0.05) respectively in bone marrow plasma from RA in comparison with OA patients were observed. In contrast, similar levels of CCL2, CXCL12 and CXCL1 in RA and OA bone marrow suggest that these cytokines do not play a significant role in the observed T cell accumulation in RA bone marrow., Conclusions: CX3CL1 and CCL5 overproduced in RA bone marrow may contribute to the accumulation of T cells observed in RA bone marrow.

Aims: Inflammatory state is considered a risk factor of atrial fibrillation (AF) occurrence. The aim of this study was a prospective evaluation of the inflammation parameters in patients with different forms of AF without structural heart disease., Methods and Results: One hundred fifty-eight patients with paroxysmal/persistent AF (87; 55.1% men, mean age 65.8±9.6 years) without structural heart disease were enrolled in the study. Inflammatory parameters: WBC, ESR, hs-CRP, IL-6, IL-15 and TNF-alpha were measured at baseline and after one year follow-up. Despite frequent AF episodes median values of WBC, ESR and C-reactive protein at baseline and after follow up were within normal ranges. There were no significant differences between WBC, ESR and hs-CRP regarding AF types. In patients who developed permanent AF form (n=14) hs-CRP concentrations were higher at baseline: 0.35 (IQR1: 0.09 IQR: 0.61) vs 0.15 (IQR1: 0.07 IQR: 0.29), p<0.01. Nevertheless, after one year's observation these differences were not significant. Among all cytokines were studied only IL-15 was significantly correlated with the number of AF episodes (r=0.26), mean (IQ1-IQ3): 10 (3-30) vs 60 (50-100), p=0.00681., Conclusion: Basic inflammatory markers were not changed in patients with refractory atrial fibrillation episodes in prospective one year's observation. Only cytokine IL-15 was correlated to numbers of AF episodes. It's potential role as a marker of arrhythmia deserves further evaluation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The pathologies of tendon and ligament attachments are called enthesopathies. Enthesitis is one of enthesopathies and it is considered a characteristic sign of rheumatic diseases from the spondyloarthritis group, including peripheral spondyloarthritis. Therefore, enthesitis has been included in a number of clinical classifications for diagnosing these diseases. Clinical diagnosis of enthesitis is based on rather non-specific clinical signs and results of laboratory tests. It is believed that imaging examinations might improve diagnosis, particularly because numerous papers prove that differentiating enthesitis from other enthesopathic processes is possible. On the other hand, a number of authors report the lack of specific signs in imaging as well as typical histological and immunological features that would enable confirmation of clinical diagnosis of enthesitis. The first part of the publication presented theories on the etiopathogenesis of enthesitis (inflammatory, mechanical, autoimmune and associated with the synovio-entheseal complex) as well as on the formation of enthesophytes (inflammatory, molecular and mechanical). This paper - the second part of the article, is a review of the state-of-the-art on the ability of imaging examinations to diagnose enthesitis. It turns out that none of the enthesitis criteria used in imaging examinations is specific for inflammation. As enthesitis may be the only symptom of early spondyloarthritis (particularly in patients with absent HLA-B27 antigen), the lack of its unambiguous picture in ultrasound and magnetic resonance imaging prompts the search for other signs characteristic of spondyloarthritis and more specific features in imaging in order to make a diagnosis as early as possible.

The pathologies of tendon and ligament attachments are called enthesopathies. One of its types is enthesitis which is a characteristic sign of peripheral spondyloarthropathy. Clinical diagnosis of enthesitis is based on rather non-specific clinical signs and results of laboratory tests. Imaging examinations are highly promising. Numerous publications prove that enthesitis can be differentiated from other enthesopathic processes in an ultrasound examination or magnetic resonance imaging. However, some reports indicate the lack of histological criteria, specific immunological changes and features in imaging examinations that would allow the clinical diagnosis of enthesitis to be confirmed. The first part of the publication presents theories on the etiopathogenesis of enthesopathies: inflammatory, mechanical, autoimmune, genetic and associated with the synovio-entheseal complex, as well as theories on the formation of enthesophytes: inflammatory, molecular and mechanical. The second part of the paper is a review of the state-of-the-art on the ability of imaging examinations to diagnose enthesitis. It indicates that none of the criteria of inflammation used in imaging medicine is specific for this pathology. As enthesitis may be the only symptom of early spondyloarthropathy (particularly in patients with absent HLA-B27 receptor), the lack of its unambiguous picture in ultrasound and magnetic resonance scans prompts the search for other signs characteristic of this disease and more specific markers in imaging in order to establish diagnosis as early as possible.

Objectives: The presence of human leukocyte antigen B27 (HLA-B27) is strongly associated with ankylosing spondylitis. HLA-B27 testing is routinely applied in the diagnosis of this disease. The aim of the present study was to compare two methods of HLA-B27 detection - a genetic sequence-based method and a flow cytometry assay., Material and Methods: Peripheral blood was obtained from 300 individuals with suspected spondyloarthropathy. Expression of HLA-B27 on the T cell surface was analysed by flow cytometry assay using GS145.2 monoclonal antibody specific for HLA-B27. DNA was isolated from the whole blood. Genes coding for HLA-B27, -B40 and -B47:01 were detected by polymerase chain reaction using the MW02/MW09 primer pair. Then, positive samples were sequenced in order to discriminate allelic variations of the HLA-B27 gene. Results of sequencing were analysed using Chromas LITE 2.1.1 software, BLAST software and the IMGT/HLA database. Ambiguous samples were additionally analysed by polymerase chain reaction using E91 and E136 primers amplifying a 135-bp fragment of the human HLA-B27 gene., Results: Among 300 samples, 76 were HLA-B27-positive on the basis of flow cytometry analysis. Genetic sequence analysis confirmed positivity of 73 from among 76 samples. Two hundred twenty six samples were HLA-B27-negative, whereas the result of one sample analysis was ambiguous. Fifty-three samples were identified as allelic variation 27:05, 19 samples as allelic variation 27:02, and one sample as allelic variation 27:07., Conclusions: This study shows that the genetic sequence-based method and the flow cytometry assay give consistent results in 99% of cases. The performed genetic analysis proves that the majority of HLA-B27-positive samples belong to the 27:05 allelic variation, which is strongly associated with high risk of ankylosing spondylitis.

For many years, it was thought that synovial cells and chondrocytes are the only sources of proinflammatory cytokines and growth factors found in the synovial fluid in patients suffering from osteoarthritis and rheumatoid arthritis. Currently, it is more and more frequently indicated that adipose tissue plays a significant role in the pathogenesis of these diseases as well as that a range of pathological processes that take place in the adipose tissue, synovial membrane and cartilage are interconnected. The adipose tissue is considered a specialized form of the connective tissue containing various types of cells which produce numerous biologically active factors. The latest studies reveal that, similarly to the synovial membrane, articular adipose tissue may take part in the local inflammatory response and affect the metabolism of the cartilage and subchondral osseous tissue. In in vitro conditions, the explants of this tissue obtained from patients suffering from osteoarthritis and rheumatoid arthritis produce similar pro- and anti-inflammatory cytokines to the explants of the synovial membrane. At this stage already, knowledge translates into imaging diagnostics. In radiological images, the shadowing of the periarticular soft tissues may not only reflect synovial membrane pathologies or joint effusion, but may also suggest inflammatory edema of the adipose tissue. On ultrasound examinations, abnormal presentation of the adipose tissue, i.e. increased echogenicity and hyperemia, may indicate its inflammation. Such images have frequently been obtained during ultrasound scanning and have been interpreted as inflammation, edema, hypertrophy or fibrosis of the adipose tissue. At present, when the knowledge concerning pathogenic mechanisms is taken into account, abnormal echogenicity and hyperemia of the adipose tissue may be considered as a proof of its inflammation. In the authors' own practice, the inflammation of the adipose tissue usually accompanies synovitis. However, we also diagnose cases in which the inflammatory process in the joint is no longer active, but abnormal vascularity still persists in the adipose tissue. There are also cases where abnormal adipose tissue is the only sign of inflammation. Therefore, ultrasound examination confirms the existence of the additional site of inflammation, i.e. the adipose tissue which should be evaluated at the stage of initial diagnosis and during follow-up, also in terms of remission.

Assessing the pathology of the synovium, its thickening and increased vascularity through ultrasound and magnetic resonance examinations (more often an ultrasound study alone) is still considered a sensitive parameter in the diagnosis of rheumatoid arthritis and in monitoring of treatment efficacy. Magnetic resonance studies showed that, aside from the joint pannus, the subchondral bone tissue constitutes an essential element in the development of rheumatoid arthritis. Bone marrow edema correlates with inflammation severity, joint destruction, clinical signs and symptoms of rheumatoid arthritis, and thus is considered a predictor of rapid radiological progression of the disease. The newest studies reveal that bone marrow edema may be a more sensitive indicator of the response to therapy than appearance of the synovium. Bone marrow edema presents with increased signal in T2-weighted images, being most visible in fat saturation or IR sequences (STIR, TIRM). On the other hand, it is hypointense and less evident in T1-weighted images. It becomes enhanced (hyperintense) after contrast administration. Histopathological studies confirmed that it is a result of bone inflammation (osteitis/osteomyelitis), i.e. replacememt of bone marrow fat by inflammatory infiltrates containing macrophages, T lymphocytes, B lymphocytes, plasma cells and osteoclasts. Bone marrow edema appears after a few weeks from occurrence of symptoms and therefore is considered an early marker of inflammation. It correlates with clinical assessment of disease activity and elevated markers of acute inflammatory phase, i.e. ESR and CRP. It is a reversible phenomenon and may become attenuated due to biological treatment. It is considered a "herald" of erosions, as the risk of their formation is 6-fold higher in sites where BME was previously noted.

Objectives: To evaluate the chondrogenic potential, phenotype and percentage of IA adipose-derived mesenchymal stem cells (ADSCs) from RA patients in comparison with OA patients. The effect of TNF treatment on ADSC differentiation was also examined., Methods: Adipose tissue was obtained from RA and OA patients. ADSCs were isolated and cultured until passage 4. After that period, the phenotype and percentage of these cells were analysed by flow cytometry. Passage 4 cells were cultured in chondrogenic medium with or without TNF. After 3 weeks of differentiation the expression of Sox9, aggrecan (Acan) and collagen 2a (Col2a) mRNA was assessed by RT-PCR and GAG deposition by alcian blue staining., Results: The phenotype and percentage of ADSCs were similar in both RA and OA. The results of alcian blue staining showed effective chondrogenesis in RA and OA ADSCs. TNF inhibited GAG deposition in both RA and OA samples similarly. Sox9, Acan and Col2a mRNA expression was significantly increased in chondrogenic-medium-treated cells (P<0.05) and decreased after TNF exposure (P<0.01). No statistically significant differences between RA and OA were observed., Conclusion: ADSCs from RA and OA patients are similar with regard to their phenotype, percentage in IA tissue and chondrogenic potential, which is reduced after exposure to TNF.

Rheumatoid arthritis is a chronic inflammatory disease with a multifactorial etiology and varied course, which in the majority of patients leads to partial disability or to permanent handicap. Its characteristic trait is a persistent inflammation of the synovial membrane and the formation of an invasive synovial tissue, called the pannus, which in time leads to destruction of the cartilage, subchondral bone tissue, and the soft tissue of the affected joint(s). The pathogenesis of rheumatoid arthritis is complex and involves cells of both innate and adaptive immunity, a network of various cytokines and an immunoregulatory dysfunction. An important role in the discovery of rheumatoid arthritis pathogenesis was played by magnetic resonance imaging, which showed the disease process to extend beyond the synovium into the bone marrow. Many studies have shown a strict correlation between the vascularity of the synovium (assessed through the power Doppler ultrasound and magnetic resonance examinations), bone marrow edema and the clinical, laboratory and histopathological parameters of rheumatoid arthritis. From the current understanding of rheumatoid arthritis, bone erosions could occur from two directions: from the joint cavity and from the bone marrow. With power Doppler ultrasound, as well as in magnetic resonance imaging, it is possible to visualize the well-vascularized pannus and its destructive effects on joint structures and ligaments. In addition, the magnetic resonance study shows inflammatory and destructive changes within the bone marrow (bone marrow edema, inflammatory cysts, and erosions). Bone marrow edema occurs in 68-75% of patients with early rheumatoid arthritis and is considered to be a predictor of rapid disease progression.

Increasing evidence suggests that enhanced stimulation of the heart and kidney by mineralocorticoids plays significant role in development of the post-infarct cardiac failure. Because increased synthesis of mineralocorticoid receptors (MR) is one of the putative factors determining pathogenic effects of mineralocorticoids we decided to determine whether the myocardial infarct results in an enhanced expression of MR mRNA and MR protein. To this end male Sprague-Dawley rats were subjected either to ligation of the left coronary artery or to sham surgery. After four weeks expressions of MR mRNA and MR protein were evaluated in both groups of rats in the left (LV) and right (RV) ventricle walls, and in the renal cortex and renal medulla by means of semiquantitative PCR and Western blotting methods. Coronary ligation resulted in the myocardial infarction encompassing 30.2% +/- 1.9% (range 23-40%) of the left ventricle wall. In the infarcted rats expression of MR mRNA was significantly greater than in the sham-operated rats, both in the LV (P<0.02) and in the RV (P<0.005). In the left but not in the right ventricle increased MR mRNA expression was associated with significant increase in expression of MR protein (P<0.001). In the renal cortex and renal medulla MR mRNA and MR protein expression in the infarcted and the sham-operated rats did not differ. The study reveals that during the post-infarct state expression of MR mRNA is elevated in both cardiac ventricles while expression of MR mRNA protein is increased only in the left ventricle. The results suggest that the enhanced expression of mineralocorticoid receptors may contribute to enhanced effects of mineralocorticoids in the heart during the post-infarct state.

Objective: To study the therapeutic potential of taurolidine (TRD), a derivative of taurine with known anti-inflammatory and anti-proliferative properties, in various experimental models of synovitis., Methods: In vitro: fibroblast-like synoviocytes (RA FLS) isolated from the synovial tissue of patients with rheumatoid arthritis (RA) were cultured in the presence of either TRD or polyvinylpyrrolidine (PVP), the pharmaceutical stabilizer of TRD, which was used as a control. Proliferation of RA FLS and cytokine (IL-6 and IL-8) release were measured. In vivo: (A). The effect of systemic TRD treatment on the development of collagen-induced arthritis (CIA) in female DBA1/J mice was investigated. Mice were treated either with intraperitoneal injections of 1 ml of 2% Taurolin Boehringer Ingelheim (TRD +PVP) or with PVP as placebo. The incidence of arthritis, myeloperoxidase (MPO) activity in periarticular tissue, as well as serum concentration of IgG specific to collagen II (IgG alphaCII) were determined. (B). The effect of intra-articular TRD treatment was studied in rabbits with antigen-induced monoarthritis (AIA). After the induction of AIA of right knees rabbits were treated either with intra-articular injections of 0.5 ml of 2% Taurolin or 0.5ml PVP ( placebo). The animals were examined for clinical signs of arthritis and diameter of joints was measured. After termination of the experiment, the arthritic knees were examined and histopathology of the joints was assessed. In addition, serum amyloid A (SAA) concentration was measured., Results: n vitro: TRD exerted cytotoxic effect on RA FLS when applied at concentrations >100 microM. TRD at non-cytotoxic concentrations, inhibited PDGF-triggered RA FLS proliferation, reduced IL-1beta - stimulated production of IL-6 and slightly decreased intracellular content of IL-8. In vivo: (A). Intraperitoneal treatment with Taurolin significantly reduced the incidence (30%) of CIA when compared to the control mice (79%). However, Taurolin failed to control the development of CIA in mice with high serum level of IgG alphaCII (>1000 U).(B). Intra-articular application of 2% Taurolin resulted in amelioration of AIA in all treated rabbits (reduced diameter of arthritic joints and smaller rise of SAA level as compared to the control animals). Histopathologic evaluation revealed pannus formation in both groups and extensive necrotic lesions of synovial tissue treated with TRD, suggesting synoviorthesis-like effect., Conclusion: Results from AIA and from in vitro RA FLS studies suggest that intra-articular administration of TRD could be used as a "pharmacological scalpel" to remove the inflamed synovium. Our data confirmed anti-inflammatory and anti-proliferative properties of TRD in all experimental models encouraging further studies which should evaluate its therapeutic potential in RA.

In this study, in an animal model of zymosan-induced peritonitis we have tested anti-inflammatory properties of Taurolidine (TRD), a synthetic derivative of taurine. In vitro, the effect of TRD and HOCl treated TRD on peritoneal macrophages was compared with that of TauCl. We report that locally administered TRD (Taurolin) shows strong anti-inflammatory properties. TRD inhibits vascular permeability increased by inflammatory stimuli; it also significantly attenuates the influx of neutrophils into the peritoneal cavity, as well as the production of pro-inflammatory cytokines (TNF-alpha, IL-6) by peritoneal exudate cells. Chlorination of TRD resulted in the formation of chloramine (TRD-Cl), as confirmed by characteristic UV spectra. Both TRD and TRD-Cl, more effectively than TauCl, inhibited the production of IL-6 by stimulated macrophages. The effect was not dependent on its well-known anti-endotoxin activity since TRD inhibited cytokine production by macrophages stimulated with either LPS or IFN-gamma. Finally, we report that anti-inflammatory activities of TRD and taurine haloamines are mediated by different mechanisms. TRD, in contrast to TauCl and TauBr, does not induce expression of HO-1, a stress inducible enzyme with strong anti-inflammatory properties.

We report that the effect of Tau-Cl on the cell fate strongly depends on the cellular context. In leukemic Jurkat cells Tau-Cl (> 200 microM) triggers mitochondrial, p53-independent apoptosis and amplifies PCD induced by anti-Fas treatment. In contrast, Tau-Cl affects RA FLS in a dose-dependent manner. At the noncytotoxic (200-400 microM) concentrations it induces: (i) p53-dependent growth arrest (Kontny et al., 2005), and (ii) Bax translocation and caspase 9 activity. Although the last events are characteristic for apoptotic state, there is not execution of RA FLS apoptosis, probably due to simultaneous inhibition of caspase 3 activity and prevention of PARP degradation. The last two events suggest an excessive ATP deprivation in Tau-Cl-treated RA FLS. At sufficiently high concentrations (> or = 500 microM) Tau-Cl causes therefore necrosis of these cells. Altogether our results suggest that Tau-Cl is able to eliminate the cells with both functional (RA FLS) and mutated (Jurkat) p53 tumor suppressor. This observation is clinically relevant because Tau-Cl is used in many animal inflammatory models and its sodium salt (used in this study) has been introduced to human therapy (Gottardi and Nagl, 2002; Teuchner et al., 2005).

The effect of arachidonic acid (AA) combined with UVA irradiation was studied in a model system mimicking phototherapy PUVA (psoralen+UVA) ex vivo in vitro. The contribution of damage to the plasma membrane by PUVA was tested on human lymphocytes derived from healthy donors. The effect of arachidonic acid (AA) combined with UVA irradiation was compared with that of a psoralen photoadduct to AA added to the culture. The adduct, obtained photochemically and purified, was characterized by NMR and MS spectrometry as a cycloadduct of psoralen to the vinylene bond of the acid (AA<>PSO). The reactions of cultured cells, manifested 20 h after treatment by changes in apoptosis and mitochondrial depolarization, were monitored by flow cytometry by tagging lymphocytes with appropriate fluorescent probes. Treatment of lymphocyte suspension within AA doses from 40 to 100 microM gradually induced a shift from Anx-V(+) (single positive cells) to late apoptotic, Anx-V(+)PI(+) (double positive cells) in a dose dependent manner. The adduct, AAPSO, induced apoptotic changes at a concentration 2-3 times higher than free AA. Combination of psoralen (1 microM ) or arachidonic acid (20-120 microM) with UVA irradiation (2-6 J/cm(2)) accelerated the plasma membrane changes in a synergic way. Preliminary studies indicated that changes in the transmembrane potential of mitochondria paralleled the apoptosis when cells were treated by AA alone. Our findings showed that UVA radiation of lymphocytes in the presence of arachidonic acid, as in the presence of psoralen, enhanced apoptosis of cells in a synergic manner. Thus, PUVA-induced apoptosis may proceed in part by a still undefined signaling pathway(s) triggered in lymphocyte membranes.

Objective: Pro-inflammatory cytokines play a critical role in the pathogenesis of RA. A natural oxidant, TauCl exerts anti-inflammatory activities. Here, the effects of Tau and TauCl on key pro-inflammatory cytokines--IL-1beta, IL-6 and TNF-alpha production by LPS-triggered peripheral blood mononuclear cells (PBMCs) isolated from RA and OA patients and healthy blood donors--were examined., Methods: PBMCs were stimulated with LPS (24 h) in the presence of Tau or TauCl (200-400 microM). Cytokine production was measured in culture supernatants (secreted) and cells lysates (cell-associated) using specific ELISAs., Results: Production of the secretedforms of IL-1beta and IL-6 was inhibited by TauCl with IC50 approximately equal to 250 microM and 300-400 microM respectively, in all investigated groups. In all cultures of PBMCs TauCl raised the TNF-alpha production at the low concentration (200 mM), while at the higher concentration (400 microM) either reduced it (55% of RA, 70% of OA patients and 55% of healthy donors) or exerted no effect (remainder of patients). Interestingly, Tau did not significantly affect any cytokine production., Conclusion: TauCl at high concentrations down-regulates pro-inflammatory cytokine production. However, the impact of TauCl on TNF-alpha production by PBMCs from RA is more limited than in cells isolated from OA patients.

Introduction: Oxidative modification of proteins affects their biological properties. Previously we have shown that hypochlorite (HOCl), the product of activated neutrophils, enhances protein immunogenecity. Collagen type II, a primary component of cartilage, is commonly used in the induction of arthritis in animals (CIA). The aim of this study was to examine whether HOCl may affect immunogenic, tolerogenic, and arthritogenic properties of collagen., Materials and Methods: DBA/J mice were injected with either native (CNAT) or chlorinated collagen (CHOCl) to induce arthritis. The effect of chlorination on collagen properties was measured by evaluation of incidence and severity of CIA. Moreover, the concentration of serum anti-collagen IgG antibodies and myeloperoxidase (MPO) activity in inflamed joints was determined., Results: Mice immunized with CNAT in adjuvant developed arthritis (CIA) with an incidence of 69%. CNAT also exerted tolerogenic properties when injected intravenously either before or shortly after primary immunization, resulting in decreased incidence and severity of CIA, reduced MPO activity in inflamed joints, and lowered serum levels of anti-CNAT IgG anti-bodies. Chlorination of collagen significantly diminished its ability to induce CIA and to trigger generation of anti-CNAT IgG antibodies. Interestingly, chlorination did not affect tolerogenic properties of collagen administered prior to primary immunization with CNAT., Conclusions: These results suggest that chlorination of collagen may selectively affect functional epitopes of collagen. It is likely that in inflamed joints, neutrophil derived HOCl, in some circumstances, will destroy arthritogenic and immunogenic B cell epitopes, while regulatory T cell epitopes will be preserved.

Vasopressin plays significant role in regulation of blood pressure by means of V1 and V2 receptors, however regulation of synthesis of these receptors in hypertension is only poorly recognized. The purpose of the present study was to compare expression of V1a, V1b and V2 vasopressin (R) mRNA in the renal cortex, renal medulla and the heart of hypertensive renin transgenic TGR(mRen2)27 rats (TGR) and of their parent normotensive Sprague Dawley (SD) strain. The study was performed on 12 weeks old TGR and SD rats. Competitive PCR method was used for quantitative analysis of V1a, V1b and V2 receptors mRNA in fragments of renal cortex, renal medulla and apex of the left ventricle of the heart. In both strains expression of V1aR and V2R mRNA was significantly greater in the renal medulla than in the renal cortex. In the renal medulla but not in the cortex expression of V1aR mRNA was significantly greater in TGR than in SD rats. V2R mRNA expression was similar in the renal cortex and renal medulla of both strains. V1aR mRNA was well expressed in the heart of SD and TGR rats, however there was no significant difference between these two strains. V2R mRNA was not present in the heart. V1bR mRNa could not be detected either in the kidney or in the heart. The results provide evidence for specific increase of expression of V1a receptors mRNA in the renal medulla of TGR rats.

The hallmarks of rheumatoid arthritis (RA) are leukocytic infiltration of the synovium and expansiveness of fibroblast-like synoviocytes (FLS). The abnormal proliferation of FLS and their resistance to apoptosis is mediated, at least in part, by present in RA joints proinflammatory cytokines and growth factors. Because IL-15 exerts properties of antiapoptotic and growth factors, and is produced by RA FLS, we hypothesized that IL-15 participates in RA FLS activation. To test this hypothesis, we first examined whether RA FLS express chains required for high affinity functional IL-15R. Indeed, RA FLS express IL-15Ralpha at mRNA and protein levels. Moreover, we confirmed the presence of IL-2Rbeta and common gamma-chains. Interestingly, TNF-alpha or IL-1beta triggered significant elevation of IL-15Ralpha chain at mRNA and protein levels. Next, we investigated the effects of exogenous or endogenous IL-15 on Bcl-2 and Bcl-x(L) expression, FLS proliferation, and apoptosis. Exogenous IL-15 enhanced RA FLS proliferation and increased the level of mRNA-encoding Bcl-x(L). To test the role of endogenous IL-15 in the activation of RA FLS, an IL-15 mutant/Fcgamma2a protein exerting properties of specific antagonist to the IL-15Ralpha chain was used. We found that blocking IL-15 biological activities using this protein substantially reduced endogenous expression of Bcl-2 and Bcl-x(L), and RA FLS proliferation that was reflected by increased apoptosis. Thus, we have demonstrated that a distinctive phenotype of RA FLS, i.e., persistent activation, proliferation, and resistance to apoptosis, is related to the autocrine activation of IL-15Rs by FLS-derived IL-15.

Objective: Taurine chloramine (Tau-Cl) has been shown to inhibit the production of proinflammatory cytokines (interleukin-6 [IL-6] and IL-8) by fibroblast-like synoviocytes (FLS) isolated from rheumatoid arthritis (RA) patients. The present study was conducted to elucidate the mechanism of inhibitory action exerted by Tau-Cl., Methods: The effects of Tau-Cl on 1) the transcription of genes coding for IL-6 and IL-8, and 2) the activity of nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1) transcription factors, which are crucial for the transcription of these cytokine genes, were investigated in FLS isolated from the synovial tissue of RA patients. FLS were cultured in vitro for 3-6 passages and stimulated with recombinant human IL-1beta (1 ng/ml) in the presence of either Tau or Tau-Cl, which were added simultaneously with the stimulus at concentrations of 250 microM or 500 microM. The relative expression of IL-6 and IL-8 messenger RNA (mRNA) was evaluated after 4 hours of stimulation, using competitive reverse transcriptase-polymerase chain reaction. The DNA binding activity of NF-kappaB and AP-1 was examined 30 minutes and 2 hours after cell stimulation, respectively, using electromobility gel shift assay., Results: IL-1beta triggered a significant rise in the activity of transcription factors NF-kappaB and AP-1, followed by an elevation of cytokine IL-6 and IL-8 mRNA expression. Tau-Cl, but not Tau, reduced IL-1beta-triggered cytokine mRNA expression, exerting stronger inhibitory activity on the levels of IL-6 than on those of IL-8. Importantly, Tau-Cl also diminished the activity of NF-kappaB and, to a lesser extent, that of AP-1 transcription factor. Neither IL-1beta nor Tau-Cl affected the activity of octamer transcription factor 1., Conclusion: Tau-Cl inhibition of IL-6 and IL-8 synthesis in FLS from RA patients results from the ability of this compound to diminish the activity of the major transcriptional regulators (NF-kappaB and AP-1), which subsequently reduces the transcription of these cytokine genes.

The implication of select protein kinase C (PKC) isoenzymes in cytokine production by human monocytes was investigated using an isozyme-selective inhibitor of PKC, rottlerin. We found that lipopolysaccharide (LPS) triggers cytosol-to-membrane translocation of PKCalpha and delta isoenzymes, whereas phorbol ester (PMA) induces translocation of several PKC isoforms. Moreover, we show that in LPS- and PMA-stimulated monocytes rottlerin affects several cellular responses. (1) At low (15 microM) concentration it blocks translocation of PKCdelta, diminishes DNA binding activity of AP-1 transcription factor, and attenuates cytokine production [tumor necrosis factor alpha (TNF-alpha) > interleukin-1beta (IL-1beta)]. (2) At high (50 microM) concentration it prevents translocation of PKCalpha, and subsequently inhibits ERK1/ERK2 phosphorylation, DNA binding activities of AP-1 and nuclear factor-KB transcription factors, and the production of both tested cytokines. Thus, we propose that cytosol-to-membrane translocation of PKCalpha and PKdelta isoenzymes may represent early steps in the signaling cascades that lead to TNF-alpha and IL-1beta production in human monocytes.

Objective: To examine whether taurine (Tau) or its physiologic chlorinated derivative, taurine chloramine (Tau-CI), affects proliferation of, and proinflammatory cytokine (interleukin-6 [IL-6] and IL-8) production by, fibroblast-like synoviocytes (FLS) isolated from rheumatoid arthritis (RA) patients., Methods: FLS, isolated from the synovial tissue of 19 RA patients and cultured in vitro for 3-6 passages, were stimulated with the recombinant human cytokines IL-1beta (1 ng/ml), tumor necrosis factor alpha (TNFalpha; 10 ng/ml), or IL-17 (10 ng/ml) in the presence of either Tau or Tau-Cl, which were added at concentrations of 50-500 microM. Tau and Tau-Cl were added simultaneously with, 2 hours before, or 24 hours after the stimuli. The concentrations of IL-6 and IL-8 were determined in culture supernatants using specific enzyme-linked immunosorbent assays. Proliferation of FLS was estimated on the basis of 3H-thymidine incorporation into the cells, which were cultured for 72 hours in the presence of recombinant human basic fibroblast growth factor (bFGF) (1 ng/ml) and Tau or Tau-Cl, which were added simultaneously at the beginning of the culture., Results: Cultured in vitro, RA FLS spontaneously secreted low levels of IL-6 and IL-8, but when RA FLS were stimulated with IL-1beta, TNFalpha, or IL-17, significantly higher amounts of IL-6 and IL-8 were produced. Tau-Cl, but not Tau, inhibited cytokine-triggered synthesis of IL-6 (50% inhibitory concentration [IC50] approximately 225 microM) and IL-8 (IC50 approximately 450 microM) when added simultaneously with the stimuli. However, IL-17-induced production of IL-8 was not affected by Tau-Cl. In the cells prestimulated with IL-1beta for 24 hours, Tau-Cl still inhibited synthesis of IL-6, but did not affect IL-8 production. Moreover, Tau-Cl inhibited spontaneous and bFGF-triggered proliferation of FLS in a dose-dependent manner. Neither Tau nor Tau-Cl affected cell viability., Conclusion: The results of these studies demonstrate that Tau-Cl inhibits production of proinflammatory cytokines by RA FLS, as well as proliferation of these cells. Thus, Tau-Cl may act as a physiologic modulator of FLS functions related to their pathogenic role in RA.

The authors hypothesized that certain PKC isoforms play an important role in the induction of pro-inflammatory cytokine (TNF-alpha, IL-1beta, IL-6) synthesis. To test this hypothesis, the cytosol-to-membrane translocation of select PKC isoforms with tested cytokine production in human monocytes cultured in vitro was correlated. It is reported that in monocytes treated with phorbol ester (PMA), translocation of PKC isoforms alpha, betaII, delta and epsilon precede cytokine synthesis. Moreover, specific inhibition of PKC translocation that occurs in the presence of Calphostin C is reflected in downstream events: lack of MAP kinases phosphorylation, loss of DNA binding ability by AP-1 transcription factor, and the reduction of pro-inflammatory cytokine synthesis. Thus, the cytosol-to-membrane translocation of PKC isoforms alpha, betaII, delta and epsilon with the subsequent activation of: (1) MAP kinases; and (2) AP-1 transcription factor, may represent critical steps in the induction of signalling cascade leading to TNF-alpha, IL-1beta, IL-6 synthesis in human monocytes., (Copyright 1999 Academic Press.)

Interleukin-12 (IL-12) is a potent immunoregulatory cytokine that exhibits antitumor activity in many experimental tumor models. In the present study, we investigated the ability of IL-15, a cytokine sharing many functions of IL-2, to modulate antitumor effectiveness of IL-12 against B16F10 melanoma in mice. In a model of locally growing tumor, intratumoral (i.t.) administration of IL-12, in three cycles of five consecutive daily injections (0.1 mug) followed by 2 days of rest, led to considerable delay of tumor development but no curative response was achieved. When combined with IL-12, subtherapeutic doses of IL-15 (0.4 mug) pontentiated the antitumor effects of IL-12 and induced complete tumor regressions in 50% of mice. Similar results were obtained in a model in which tumor-bearing mice were intravenously co-injected with melanoma cells to induce metastases. Combined administration of IL-12 and IL-15 yielded greater antitumor activity than injections of either cytokine alone and resulted in prolonged survival of mice bearing locally growing tumor and metastases. Studies of immunological parameters in mice treated with both IL-12 and IL-15 have shown enhanced NK activity (against YAC-1 cells) in the spleen and stimulation of both NK activity and specific anti-B16F10 cytotoxic effector cells in tumor-draining lymph nodes (LN). The strong antitumor effect of the IL-12 + IL-15 combination correlated with a high serum level of IFN-gamma in the treated mice. Moreover, increased expression of IL-15Ralpha was demonstrated in LN lymphocytes isolated from mice injected with IL-12. This result together with findings of other authors showing enhanced expression of IL-12 receptor by IL-15 [1] suggests that the augmentation of the antitumor effect during the course of IL-12/IL-15-based therapy could result from reciprocal upregulation of receptors by both cytokines and synergistic effects on IFN-gamma induction.

It is known that lymphocytes express functional muscarinic cholinergic receptors. In this study, RT-PCR method was applied to study the presence and relative levels of mRNA encoding muscarinic receptor subtypes in human peripheral blood mononuclear cells (PBMCs). Our results, confirmed by DNA sequencing, demonstrate the presence of m2, m3, m4, and m5 receptor subtypes in human PBMCs. The relative levels of muscarinic receptor subtypes fit the following pattern: m3 > m5 > m4 > m2. Our data provide strong evidence confirming previous pharmacological studies that suggested the existence of several subtypes of muscarinic receptors on human PBMCs. We cannot exclude the possibility that expression of receptor subtype depends on the lineage and/or activation status of the cell.

Kinetic and equilibrium studies of [3H]QNB and [3H]nicotine binding to human peripheral blood lymphocytes have been performed. The calculated number of muscarinic binding sites is 6 x 10(4) per cell, and of nicotinic binding sites is 2 x 10(3) per cell. The conditions for routine estimations of muscarinic and nicotinic receptors on human lymphocytes have been established. Significant differences in the distribution of muscarinic and nicotinic binding sites among normal PBL and malignant cell lines have been described.

The muscarinic acetylcholine receptors on rat lymphocytes were determined by [3H]quinuclidinyl benzilate binding studies. Binding of [3H]quinuclidinyl benzilate is rapid (half saturation occurred within 120 s) and highly specific. Muscarinic receptors reveal high lability. The number of receptors on plasma membrane depends on time of incubation as well as on composition of incubation medium. Lymphocytes incubated in nutrient-deficient media lose their surface receptors; enrichment of the medium causes reappearance of the receptors. Appearance of [3H]quinuclidinyl benzilate-binding sites in the incubation medium was under conditions in which binding to lymphocytes was decreased. It is concluded that the number of plasma membrane receptors on rat lymphocytes represents the dynamic steady state in which newly synthesized and degraded receptors are balanced.

In the light of the data presented, the previously described explanation of the unusual shape of the kinetic curve of the muscarinic antagonist 3H-QNB binding to rat lymphocytes, with apparent maximum of bound radioactivity after 5 min of incubation, followed by decrease of specific binding, is not satisfactory. The kinetics of this binding carried out at 4 degrees C, 20 degrees C and 37 degrees C, or in the presence of the metabolic poison sodium azide, suggests involvement of an energy-consuming mechanism in this process. The surface receptors, sensitive to bacterial protease degradation before 3H-QNB binding, become somehow resistant to this protease action after 5 min of incubation of the cells with 3H-QNB. A possible explanation for this phenomenon, based on internalization in cell endosomes, is presented.

In unstimulated lymphocytes acetylcholine caused concentration-dependent increase of RNA synthesis. Atropine and tubocurarine did not inhibit this stimulating effect, but, on the contrary, gave an additional peak of uridine incorporation. In lymphocytes stimulated previously with PHA, acetylcholine produced changes in RNA synthesis which did not depend on the dose of the mitogen. The stimulating effect of acetylcholine was inhibited at all concentrations by tubocurarine, while atropine abolished only the positive effects of higher acetylcholine concentrations. The results suggest occurrence of three different types of acetylcholine action on rat lymphocytes.

Thymocytes at various stages of their ontogeny have been studied in relation to their ability to bind [3H]quinuclidinyl benzilate [( 3H]QNB), a specific radioligand of the muscarinic cholinergic receptors. [3H]QNB-specific binding to thymocytes from 15-19-day fetal, newborn and adult thymuses of mice and rats was compared and correlated. Our experiments showed that the kinetics of [3H]QNB binding to thymocytes at 37 degrees C was similar to that of the lymph node lymphocytes (LNL) with maximum after 5 min of incubation and subsequent decrease to 10% of the maximum after 90 min of incubation. Maximal binding for the entire thymocyte population was twice lower than for the cortisone-resistant thymocytes (CRT) or for LNL. Binding of [3H]QNB carried out at 4 degrees C resulted in disappearance of the maximum, but did not alter the difference between CRT and entire thymocyte population. Depletion of CRT detectable [3H]QNB-specific binding to thymocytes until 18th day of gestation but the maximal binding increased up to 20% at the day 19 and reached 90% of adult level on the third day after birth. Moreover, carbamylcholine (a muscarinic agonist) treatment in vivo induced a significant decrease in [3H]QNB binding to the thymocytes. We thus suggest that a subpopulation of thymocytes bearing muscarinic receptors in the periphery acquired these receptors in the thymus as one of the last steps of their maturation. We cannot exclude the possibility that cholinergic stimulation might trigger these lymphocytes to leave the thymus.

The conditions of the binding of acetylcholine have been studied in lymphocytes isolated from rat peripheral lymph nodes. Acetylcholine appeared to penetrate the lymphocyte membrane. We have confirmed the presence of muscarinic receptors, which, however, are not involved in transport of acetylcholine through the membrane. The receptors of the nicotine type on lymphocytes are demonstrated by the decrease of acetylcholine binding in the presence of a specific antagonist, tubocurarine. These nicotinic receptors may be involved in acetylcholine transport into the cells.

Muscarinic receptors were studied on lymphocytes from young and old Wistar rats. Binding studies were performed by the use of [3H]-QNB, a specific muscarinic antagonist. Some differences between these two groups were observed. Maximal binding of [3H]-QNB and half time of the maximal binding is lower for lymphocytes of old rats [3H]-QNB receptor complexes could not be found in the supernatants derived from lymphocytes of old animals. Higher ability to loose or hide the muscarinic receptors was also observed in this group of rats. All these observations could reflect a more effective degradation, as well as a lower level of muscarinic receptors exposed on lymphocytes from old animals.

The level of muscarinic receptors on lymphocytes from rheumatoid arthritis (RA) patients in comparison with healthy individuals was determined by the binding studies of a specific muscarinic ligand [3H]-QNB in the presence of the competitive antagonist atropine. The response to phytohaemagglutinin and the influence of acetylcholine on this process in patients' lymphocytes was also studied. We have found direct correlation among PHA stimulation indices, the level of muscarinic receptors, and the influence of acetylcholine on PHA stimulation indices. Differences in these parameters occurring in systemic forms of RA as compared to other RA patients are also described and their possible mechanism discussed. The influence of cholinergic stimulation on the reactivity of lymphocytes after PHA in different forms of RA could depend on the number of muscarinic receptors present on the lymphocyte surface, as well as on the activity of soluble factors, which could modulate the functional state of the receptors.