Flemish network on rare connective tissue diseases (CTD): patient pathways in systemic sclerosis. First steps taken.

Despite the low prevalence of each rare disease, the total burden is high. Patients with rare diseases encounter numerous barriers, including delayed diagnosis and limited access to high-quality treatments. In order to tackle these challenges, the European Commission launched the European Reference Networks (ERNs), cross-border networks of healthcare providers and patients representatives. In parallel, the aims and structure of these ERNs were translated at the federal and regional levels, resulting in the creation of the Flemish Network of Rare Diseases. In line with the mission of the ERNs and to ensure equal access to care, we describe as first patient pathways for systemic sclerosis (SSc), as a pilot model for other rare connective and musculoskeletal diseases. Consensus was reached on following key messages: 1. Patients with SSc should have multidisciplinary clinical and investigational evaluations in a tertiary reference expert centre at baseline, and subsequently every three to 5 years. Intermediately, a yearly clinical evaluation should be provided in the reference centre, whilst SSc technical evaluations are permissionably executed in a centre that follows SSc-specific clinical practice guidelines. In between, monitoring can take place in secondary care units, under the condition that qualitative examinations and care including interactive multidisciplinary consultations can be provided. 2. Patients with early diffuse cutaneous SSc, (progressive) interstitial lung disease and/or pulmonary arterial hypertension should undergo regular evaluations in specialised tertiary care reference institutions. 3. Monitoring of patients with progressive interstitial lung disease and/or pulmonary (arterial) hypertension will be done in agreement with experts of ERN LUNG.

Systematic review of international clinical guidelines for the promotion of physical activity for the primary prevention of cardiovascular diseases.

BACKGROUND: Cardiovascular diseases are the world's leading cause of morbidity and mortality. An active lifestyle is one of the cornerstones in the primary prevention of cardiovascular disease. An initial step in guiding primary prevention programs is to refer to clinical guidelines. We aimed to systematically review clinical practice guidelines on primary prevention of cardiovascular disease and their recommendations regarding physical activity. METHODS: We systematically searched Trip Medical Database, PubMed and Guidelines International Network from January 2012 up to December 2020 using the following search strings: 'cardiovascular disease', 'prevention', combined with specific cardiovascular disease risk factors. The identified records were screened for relevance and content. We methodologically assessed the selected guidelines using the AGREE II tool. Recommendations were summarized using a consensus-developed extraction form. RESULTS: After screening, 27 clinical practice guidelines were included, all of which were developed in Western countries and showed consistent rigor of development. Guidelines were consistent about the benefit of regular, moderate-intensity, aerobic physical activity. However, recommendations on strategies to achieve and sustain behavior change varied. Multicomponent interventions, comprising education, counseling and self-management support, are recommended to be delivered by various providers in primary health care or community settings. Guidelines advise to embed patient-centered care and behavioral change techniques in prevention programs. CONCLUSIONS: Current clinical practice guidelines recommend similar PA lifestyle advice and propose various delivery models to be considered in the design of such interventions. Guidelines identify a gap in evidence on the implementation of these recommendations into practice.

Multicentric reticulohistiocytosis associated arthritis responding to anti-TNF and methotrexate.

We present a patient with therapy resistant multicentric reticulohistiocytosis (MRH). MRH is a rare granulomatous, multisystem disease characterised most frequently by disfiguring papulonodular skin lesions and sometimes a destructive polyarthritis, though any organ can be involved. Abnormal histiocytic reactions to an undetermined stimulus (possibly an associated mycobacterial infection, auto immune process or neoplastic process) have been proposed as an underlying mechanism. The diagnosis is confirmed by histopathology of the cutaneous nodules and/or synovial membrane by the presence of CD68-positive histiocytes and multinucleated giant cells with an eosinophilic 'ground-glass' cytoplasm. Recent studies have identified TNFalpha and other inflammatory cytokines to be highly expressed in the synovium and synovial fluid of affected joints in patients with MRH. Based on these findings, we treated our patient with infliximab in combination with methotrexate with marked improvement of morning stiffness, tender and swollen joint count, visual analogue scale and health assessment questionnaire after his third infusion. However, the nodules did not markedly resolve. When treating patients with MRH with TNFa neutralizing drugs, one has to keep the possible association with malignancy in 15-30% of cases in mind and these products should be used with caution.

T cell signal transducer and activator of transcription (STAT) 4 and 6 are affected by adalimumab therapy in rheumatoid arthritis.

OBJECTIVES: TNF-alpha inhibition therapy affects the systemic immune response in rheumatoid arthritis by influencing T cell subtypes (Th1, Th2, Treg), but its effect on the intracellular signal transduction in T cells remains largely unexplored. Here we studied the activation of Th1-associated signalling molecule STAT4 and Th2-associated STAT6 in CD4+ T cells. METHODS: Eight rheumatoid arthritis patients were studied before and after 12 weeks of adalimumab therapy and compared to 8 healthy individuals. Peripheral blood mononuclear cells (PBMC) were analysed flow cytometrically either directly after isolation or after 24 hours of anti-CD3/anti-CD28 stimulation, to determine spontaneous and IL-4/IL-12-induced STAT4 and STAT6 phosphorylation in CD4+ T cells. Cytokine production by stimulated PBMC was measured in the supernatant using a cytometric bead array. Non-parametric statistical tests were applied. RESULTS: After adalimumab therapy, phospho-STAT6 increased, both in freshly isolated and anti-CD3/anti-CD28-stimulated CD4+ T cells. The STAT6 response to brief IL-4 stimulation did not change. In healthy individuals and adalimumab-treated patients, anti-CD3/anti-CD28 induced the phosphorylation of STAT4, but not in untreated patients. IFN-gamma production in untreated patients was significantly lower than in healthy individuals or adalimumab-treated patients. In contrast, the production of IL-4, IL-6 and IL-12 was not influenced. CONCLUSIONS: Adalimumab therapy increases Th2-associated STAT6 phosphorylation and restores the activation-induced STAT4 phosphorylation to the levels in healthy individuals. This advocates against a pro-inflammatory effect of Th1-associated STAT4 and might provide an explanation for the influence of TNF inhibition therapy on the systemic T cell response in rheumatoid arthritis.

Basophil activation test by flow cytometry: present and future applications in allergology.

The diagnosis of allergic reactions in clinical practice rests upon both clinical history and the demonstration of specific immunoglobulin E (sIgE), either in the serum or via skin tests. However, for various reasons, identification of the offending allergen(s) is not always possible. Moreover, not all allergies are IgE-mediated. In an attempt to find reliable methods to investigate hypersensitivity reactions, histamine and sulfidoleukotriene release tests have long been introduced. However, relatively few comprehensive quality reports have been published so far. Upon challenge with a specific allergen, basophils not only secrete quantifiable bioactive mediators but also upregulate the expression of different markers which can be detected efficiently by flow cytometry using specific monoclonal antibodies. This review addresses the principals, particular technical aspects and pitfalls as well as the clinical and research applications of flow-assisted analysis of in vitro activated basophils.

Combined analysis of intracellular signalling and immunophenotype of human peripheral blood basophils by flow cytometry: a proof of concept.

BACKGROUND: The signal transduction pathways and control mechanisms involved in IgE-mediated basophil activation remain incompletely understood. OBJECTIVES: To investigate whether basophilic intracellular signal transduction and immunophenotype can be analysed simultaneously by flow cytometry. METHODS: Basophils in whole blood were stimulated with anti-IgE and latex antigen at various concentrations and during different time courses. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) as a representative of the intracellular signal transduction pathway and surface expression of CD63 was assessed simultaneously flow cytometrically. The effect of pre-incubation with IL-3 was assessed. RESULTS: Stimulation of the basophils with anti-IgE and allergen induces a rapid phosphorylation of p38 MAPK that peaks between 1 and 5 min and returns to baseline levels after 60 min. In contrast, CD63 up-regulation demonstrates a maximal but more continuous expression that peaks approximately 5 min later than phosphorylation of p38 MAPK. Specific inhibition of p38 MAPK reduced or almost completely abrogated up-regulation of CD63. Pre-incubation of the basophils with IL-3 produces a rapid p38 MAPK phosphorylation over basal levels, but this was weaker and shorter than for anti-IgE stimulation. Pre-incubation of the basophils with IL-3 did not potentiate anti-IgE-induced phosphorylation of p38 MAPK and did affect spontaneous or IgE-mediated CD63 up-regulation. CONCLUSIONS: This study provides the proof that the flow cytometer allows an integrated analysis of basophilic intracellular signalling and immunophenotyping. Owing to its technical simplicity, the low number of cells required and rapid analysis, the technique seems promising for use in the clinic as a diagnostic tool or to monitor therapy. CAPSULE SUMMARY: This study is the first to provide evidence for a combined analysis of basophilic intracellular signalling and immunophenotyping by flow cytometry. Owing to its technical simplicity, the low number of cells required and rapid analysis, the technique seems promising for use in the clinic as a diagnostic tool or to monitor therapy.

Influence of anti-tumor necrosis factor therapy (Adalimumab) on regulatory T cells and dendritic cells in rheumatoid arthritis.

OBJECTIVE: To investigate whether anti-TNF therapy could have an effect on dendritic cells (DCs) and regulatory T cells in rheumatoid arthritis (RA) patients. METHODS: A four-colour flow cytometric technique was used to measure CD4+CD25+ T cells i.e. CD4+CD25high+ (regulatory T cells) and CD4+CD25low+ (activated T cells)), DCs as well as the in vitro, intracellular, lipopolysaccharide-stimulated cytokine production of DCs. RESULTS: Clinical and laboratory parameters of disease activity decreased after anti-TNF treatment. Before anti-TNF therapy, RA patients demonstrated a decreased count of Th2-promoting lymphoid DCs as compared to controls and after anti-TNF therapy this decrease was sustained. Intracellular cytokine production was only found in the myeloid DCs population and there was a higher production of TNF-alpha and IL1-b as compared to healthy controls. Treatment did not alter this cytokine production. Before anti-TNF therapy, the percentage CD4+CD25+ T cells was significantly elevated in RA patients than in healthy controls. CONCLUSION: These results demonstrate anti-TNF to be a potent anti-inflammatory drug, as mirrored by the decrease in clinical and biological parameters as well as the decrease in activated CD4+ T cells. However, in this study no demonstrable effect on DCs and regulatory T cells was found.

Antagonism of meta-chlorophenylpiperazine-induced inhibition of exploratory activity in an emergence procedure, the open field test, in rats.

The effects of meta-chlorophenylpiperazine (mCPP) were studied on exploratory behaviour in the open field test, using a procedure designed to evaluate the emergence of rats into a novel environment. mCPP reduced the exploratory activity in a dose-related manner after subcutaneous (s.c.), intraperitoneal (i.p.) and intravenous (i.v.) administration. The inhibition was manifest in all the parameters used to quantify the exploration of the open field area. Additional neuroendocrine experiments in a parallel group of rats revealed a dose-related increase in plasma prolactin and ACTH levels after i.v. mCPP, pointing to a general state of arousal in these mCPP-treated animals. A number of 5-HT antagonists were tested for their ability to prevent or reverse the behavioural inhibition induced by an i.v. injection of 1.0 g/kg mCPP given 15 min before testing in the open field. The antagonists were injected s.c. or given orally at various time intervals before mCPP, or they were injected i.v. 10 min after mCPP. The lowest active doses for the attentuation of the mCPP-induced behavioural inhibition after s.c., oral and i.v. administration, respectively, were 0.04, 40 and 10 mg/kg for pizotifen; 0.16, 0.16 and 0.16 mg/kg for mianserin; 0.63, 0.16 and 0.16 mg/kg for methysergide, and 0.16, 2.5 and 2.5 mg/kg for ritanserin. The lowest active doses of mirtazapine after s.c. and i.v. treatment were 0.01 and 0.16 mg/kg. These data indicate that mixed 5-HT1/5-HT2 receptor antagonists such as pizotifen and methysergide, and mixed 5-HT and catecholamine antagonists such as mianserin and mirtazapine are more potent antagonists of mCPP-induced behavioural inhibition in rats than the more selective 5-HT2A/5-HT2C antagonist ritanserin.

Antagonism of the discriminative stimulus properties of cocaine with the combination of a dopamine D1 and D2 antagonist.

Antagonism of the discriminative stimulus properties of 10 mg/kg cocaine was studied in rats by use of the dopamine D1 antagonist SCH 23390 and the D2 antagonist haloperidol. Whereas SCH 23390 and haloperidol were by themselves unable to antagonize the cueing properties of cocaine, the combination of both dopamine antagonists resulted in a complete blockade of the cocaine cue. In the presence of a fixed dose of 0.01 and 0.04 mg/kg haloperidol, the ED50's (it is the effective dose in 50% of the animals) of SCH 23390 for cocaine antagonism were 0.043 and 0.012 mg/kg, respectively. Similarly, the ED50's of haloperidol in combination with 0.01 and 0.04 mg/kg SCH 23390 were 0.021 and 0.024 mg/kg. The combined treatment of haloperidol and SCH 23390 resulted in strong response-rate reductions. At all combination regimens resulting in a complete blockade of the cocaine cue, response rate was reduced to less than 20% of the control values. These results indicate that the cueing properties of cocaine are both dopamine D1- and D2-mediated and that a combined antagonism of both receptor subtypes can lead to a complete antagonism of the cueing properties of cocaine which is associated with severe attenuation of response rate.

Antigenic components of Taenia saginata and their relevance to the diagnosis of bovine cysticercosis by immunoelectrophoresis.

22 antigenic components of Taenia saginata proglottides were detected by immunoelectrophoretic (IEP) study. Cross-absorption of the hyperimmune serum, raised in rabbits against T. saginata, with heterologous and host antigens showed that two thirds of these components were not specific for the cestode. Among the calves experimentally infected with T. saginata eggs, 3 precipitation patterns in IEP were identified depending on the evolutionary stage of the infection. The hydrosoluble extract of T. saginata was used as antigen. A short precipitating arc present near the antigen well and appearing 3 to 4 weeks post-infection of the calves and a long precipitating arc extending towards the anodic end of the slide and appearing in later stages of infection were found of high diagnostic value. No false-positive reaction or cross-reaction were found. Among the calves harbouring less than 50 cysticerci, false-negative reactions were seen.