[Interprofessional Care of Patients with Rheumatoid Arthritis from the Perspective of a General Practitioner Perspective: Results of a Cross-sectional Study]. / Interprofessionelle Versorgung von Patienten mit rheumatoider Arthritis aus hausärztlicher Sicht: Ergebnisse einer Querschnittserhebung.

BACKGROUND: General practitioners, specialists, occupational and physical therapists, nursing services and other professional groups are all involved in the treatment of patients with rheumatoid arthritis. This study aims to describe interprofessional cooperation in daily ambulatory care from the perspective of a general practitioner. METHODS: The cross-sectional study investigated cooperation between general practitioners (n=121 in 68 medical practices) and several other health care providers in Hesse and Rhineland Palatinate, Germany, from February to September 2017. It was part of the prospective cohort study PANORA (Prevalence of anti-cyclic citrullinated peptide (anti-CCP) positivity in patients with new onset of non-specific musculoskeletal symptoms). The questionnaire that was used contained closed-ended questions on socio-demographics and frequency of contact, and asked physicians to assess and weigh existing collaboration. Descriptive statistics were used for data analysis. RESULTS: When caring for patients with rheumatoid arthritis, 70%, of the physicians often took responsibility for synchronizing medications, and discussing diagnoses and test results. The most frequent cooperation was with rheumatologists and was considered as highly important but the least satisfactory. The second most frequent cooperation was with physical therapists and this was also rated as very important. Physicians had highest level of satisfaction with their collaboration with the nursing services. CONCLUSION: This study shows that general practitioners perform several medical tasks when treating patients with rheumatoid arthritis. During the process, they work together with several health care providers to various degrees. Cooperation with rheumatologists and physical therapists is particularly important to general practitioners; cooperation with rheumatologists is considered inadequate and in need of improvement.

High impact of COVID-19 in long-term care facilities, suggestion for monitoring in the EU/EEA, May 2020.

Residents in long-term care facilities (LTCF) are a vulnerable population group. Coronavirus disease (COVID-19)-related deaths in LTCF residents represent 30-60% of all COVID-19 deaths in many European countries. This situation demands that countries implement local and national testing, infection prevention and control, and monitoring programmes for COVID-19 in LTCF in order to identify clusters early, decrease the spread within and between facilities and reduce the size and severity of outbreaks.

Mouse basophils reside in extracellular matrix-enriched bone marrow niches which control their motility.

Basophils co-express FcεRIα and CD49b, the α-2 chain of integrin-type receptor VLA-2 (α2ß1), which recognizes type-1 collagen as a major natural ligand. The physiological relevance of this integrin for interactions with extracellular bone marrow matrix remains unknown. Herein, we examined the expression of several receptors of this family by bone marrow-derived basophils sorted either ex-vivo or after culture with IL-3. Having established that both populations display CD49d, CD49e and CD49f (α-4, α-5 and α-6 integrins subunits, respectively), we addressed receptor functions by measuring migration, adhesion, proliferation and survival after interacting with matched natural ligands. Type I collagen, laminin and fibronectin promoted basophil migration/adhesion, the former being the most effective. None of these ligands affected basophil viability and expansion. Interactions between basophils and extracellular matrix are likely to play a role in situ, as supported by confocal 3D cell imaging of femoral bone marrow sections, which revealed basophils exclusively in type-1 collagen-enriched niches that contained likewise laminin and fibronectin. This is the first evidence for a structure/function relationship between basophils and extracellular matrix proteins inside the mouse bone marrow.

Rapamycin combined with TGF-ß converts human invariant NKT cells into suppressive Foxp3+ regulatory cells.

Invariant NKT (iNKT) cells constitute a versatile T cell subset with important regulatory functions, which are thought to result essentially from their capacity to promptly produce cytokines that influence the Th1/Th2 balance. In this study, we report that these cells can also express Foxp3, an important transcriptional regulator associated with suppressive activity, once they have been exposed to TGF-ß. Foxp3 was expressed by iNKT cells from both peripheral and cord blood. CD4(+) iNKT cells acquired Foxp3 expression preferentially, although a lower proportion of their CD4(-) counterpart also became positive. All Foxp3(+) iNKT cells displayed CD25 but not necessarily CTLA4 or GITR, regardless of the upregulation of these markers in the presence of TGF-ß. Exposure to TGF-ß decreased IL-4 and IFN-γ production while increasing IL-10, independently from Foxp3 expression. IL-17 was not detected. TGF-ß induced high levels of Foxp3, but no suppressor activity, which emerged only in the presence of rapamycin. Peripheral and cord blood Foxp3(+) iNKT cells suppressed the proliferation of conventional autologous and heterologous CD4(+) T cells equally, in a cell contact-dependent and Ag-independent manner. Our findings demonstrate that human iNKT cells become suppressive in the presence of TGF-ß plus rapamycin, thus adding a new facet to their complex functional properties.

Critical role of the neutrophil-associated high-affinity receptor for IgE in the pathogenesis of experimental cerebral malaria.

The role of the IgE-FcεRI complex in malaria severity in Plasmodium falciparum-hosting patients is unknown. We demonstrate that mice genetically deficient for the high-affinity receptor for IgE (FcεRIα-KO) or for IgE (IgE-KO) are less susceptible to experimental cerebral malaria (ECM) after infection with Plasmodium berghei (PbANKA). Mast cells and basophils, which are the classical IgE-expressing effector cells, are not involved in disease as mast cell-deficient and basophil-depleted mice developed a disease similar to wild-type mice. However, we show the emergence of an FcεRI(+) neutrophil population, which is not observed in mice hosting a non-ECM-inducing PbNK65 parasite strain. Depletion of this FcεRI(+) neutrophil population prevents ECM, whereas transfer of this population into FcεRIα-KO mice restores ECM susceptibility. FcεRI(+) neutrophils preferentially home to the brain and induce elevated levels of proinflammatory cytokines. These data define a new pathogenic mechanism of ECM and implicate an FcεRI-expressing neutrophil subpopulation in malaria disease severity.

Ineffective erythropoiesis with reduced red blood cell survival in serotonin-deficient mice.

Serotonin (5-HT) has long been recognized as a neurotransmitter in the central nervous system, where it modulates a variety of behavioral functions. Availability of 5-HT depends on the expression of the enzyme tryptophan hydroxylase (TPH), and the recent discovery of a dual system for 5-HT synthesis in the brain (TPH2) and periphery (TPH1) has renewed interest in studying the potential functions played by 5-HT in nonnervous tissues. Moreover, characterization of the TPH1 knockout mouse model (TPH1(-/-)) led to the identification of unsuspected roles for peripheral 5-HT, revealing the importance of this monoamine in regulating key physiological functions outside the brain. Here, we present in vivo data showing that mice deficient in peripheral 5-HT display morphological and cellular features of ineffective erythropoiesis. The central event occurs in the bone marrow where the absence of 5-HT hampers progression of erythroid precursors expressing 5-HT(2A) and 5-HT(2B) receptors toward terminal differentiation. In addition, red blood cells from 5-HT-deficient mice are more sensitive to macrophage phagocytosis and have a shortened in vivo half-life. The combination of these two defects causes TPH1(-/-) animals to develop a phenotype of macrocytic anemia. Direct evidence for a 5-HT effect on erythroid precursors is provided by supplementation of the culture medium with 5-HT that increases the proliferative capacity of both 5-HT-deficient and normal cells. Our thorough analysis of TPH1(-/-) mice provides a unique model of morphological and functional aberrations of erythropoiesis and identifies 5-HT as a key factor for red blood cell production and survival.

Downregulation of basophil-derived IL-4 and in vivo T(H)2 IgE responses by serotonin and other organic cation transporter 3 ligands.

BACKGROUND: Murine basophils can contribute to the T(H)2 polarization of the immune response by providing rapidly large amounts of IL-4, which suggests that pharmacologic downregulation of this cytokine might provide a strategy to attenuate pathologies associated with excessive production. OBJECTIVE: We examined a number of physiological and pharmacologic ligands of the organic cation transporter 3 (OCT3), a membrane carrier of biogenic amines, for their inhibitory effect on IL-4 production by basophils, selecting the most efficient compounds for in vivo evaluation in basophil-dependent experimental models. METHODS: IL-4 production by basophils isolated ex vivo or from bone marrow cultures was assessed in response to various stimuli with or without biogenic monoamines or pharmacologic analogs. Selected compounds were administered in vivo to examine their effect on levels of circulating IgE generated during a basophil-dependent T(H)2 response and on basophil activation in mice receiving IL-33. RESULTS: We found a drastic decrease in IL-4 production by stimulated basophils on exposure to serotonin (5-hydroxytryptamine [5-HT]) that is taken up by basophils through the specific high-affinity transporters serotonin transporter and the polyspecific, high-capacity organic cation transporter 3 (OCT3; or Slc22a3) but inhibits their function exclusively through the latter. This downregulation is likewise observed in vivo in response to 5-HT and other OCT3 ligands, as well as in human basophils sorted from PMBCs of nonatopic donors. CONCLUSIONS: We provide evidence for a new means of downregulating IL-4 production by basophils, both in vitro and in vivo, through OCT3 targeted by 5-HT and pharmacologic ligands.

Proinflammatory environment dictates the IL-17-producing capacity of human invariant NKT cells.

CD1d-reactive invariant NKT (iNKT) cells have been implicated in a number of experimental models of human pathologies. Given the scope of their immunoregulatory activities mediated through distinct cytokine patterns, it has been proposed that this functional diversity originates from distinct iNKT subpopulations. In this study, we report that human CD161(+) iNKT cells are intrinsically endowed with the capacity to generate IL-17, but require TGF-ß, IL-1ß, and IL-23 to carry out this potential. IL-17-producing iNKT cells are already present in cord blood but, in contrast to peripheral blood iNKT cells, they cannot generate IFN-γ. These IL-17 producers respond to aryl hydrocarbon receptor stimulation and express IL-23 receptor and retinoic acid-related orphan receptor C, similar to conventional T helper 17 cells, from which they differ by their restricted ability to coproduce IL-22. In conclusion, IL-17 production by human iNKT cells depends on two critical parameters, namely an intrinsic program and a proinflammatory environment.

Treatment with the TLR7 agonist R848 induces regulatory T-cell-mediated suppression of established asthma symptoms.

The evolution of allergic asthma is tightly controlled by effector and regulatory cells, as well as cytokines such as IL-10 and/or TGF-ß, and it is widely acknowledged that environmental exposure to allergens and infectious agents can influence these processes. In this context, the recognition of pathogen-associated motifs, which trigger TLR activation pathways, plays a critical role with important consequences for disease progression and outcome. We addressed the question whether the TLR7 ligand resiquimod (R848), which has been shown to be protective in several experimental allergic asthma protocols, can also suppress typical asthma symptoms once the disease is established. To this end, we used an OVA-induced experimental model of murine allergic asthma in which R848 was injected after a series of challenges with aerosolized OVA. We found that the treatment attenuated allergic symptoms through a mechanism that required Tregs, as assessed by the expansion of this population in the lungs of mice having received R848, and the loss of R848-mediated suppression of allergic responses after in vivo Treg depletion. IL-10 provided only a minor contribution to this suppressive effect that was largely mediated through a TGF-ß-dependent pathway, a finding that opens new therapeutic opportunities for the pharmacological targeting of Tregs.

A natural protective function of invariant NKT cells in a mouse model of innate-cell-driven lung inflammation.

Activation of invariant natural killer T (iNKT) cells by treatment with their α-galactosyl ceramide ligand provides therapeutic benefits in several immune inflammatory settings. Given the artificial nature of this stimulation, the natural regulatory functions of iNKT remain uncertain. Addressing this issue in a mouse model of innate-cell-driven lung inflammation induced by the cytokine/alarmin IL-33 that targets iNKT cells, we found that eosinophil and neutrophil recruitment was markedly increased in treated iNKT cell-deficient (Jα18 KO) mice, as was the local production of eotaxin and keratinocyte chemoattractant chemokines. By contrast, lung inflammation decreased after adoptive transfer of iNKT cells, which restored the WT inflammatory response in Jα18 KO mice. Finally, we established that this natural anti-inflammatory function of iNKT cells depends on their IFN-γ production and on endogenous IL-12. Our study provides the first evidence of a protective role of iNKT cells during lung inflammation that does not require pharmacological TCR engagement.

Modulation of hematopoiesis through histamine receptor signaling.

Histamine is one of the most versatile biogenic amines targeting a variety of cells through extra- and intracellular binding sites and specific receptors, which trigger different signal transduction pathways. It has been associated with cell growth ever since G. Kahlson demonstrated that its synthesis was increased in rapidly growing tissues of plants and animals. He proposed that the newly formed amine, as opposed to its stored counterpart, might play a major role in growth processes. Later on, a number of investigators provided evidence for the contribution of histamine to the expansion of normal and malignant cells, whether of hematopoietic origin or not. These studies have generated conflicting results, revealing growth-promoting as well as inhibitory effects, most likely because the final outcome of exposure to histamine depends on the signaling pathways triggered by distinct receptors and their differential distribution among the target population. The purpose of the present review is to outline our current understanding of the regulatory functions of histamine during growth and differentiation of hematopoietic progenitors, focusing on those mediated through its H4 receptor.

Migration of renal tumor cells depends on dephosphorylation of Shc by PTEN.

The tumor suppressor PTEN is a phosphatase using FAK and Shc as direct substrates, and Akt as a key effector via PIP3. PTEN regulates cell migration and may influence metastases. We quantified PTEN in 135 clear cell renal cell carcinomas (ccRCC) by Western blot analysis and found statistically significant lower PTEN expression in patients who died, usually caused by metastases, within 5 years after surgery, compared to those surviving this time period. In athymic mice, PTEN transfected 786-O cells were injected into the tail vein and metastatic load of the lungs was quantified. We observed a strongly reduced metastatic load after PTEN transfection. For analyses of the PTEN activities, transfections with mutated PTEN genes were performed, leading to loss of lipid phosphatase activity and/or protein phosphatase activity, and of the C-terminal tail. Cell migration was analyzed in a Boyden chamber and phosphorylation of PTEN downstream targets Akt, FAK and Shc by Western blotting. 786-O cells transfected with the functional PTEN gene showed profoundly diminished migration. Transfection with a mutated PTEN isoform leading to loss of protein phosphatase activity, but not of lipid phosphatase activity, abolished this effect. Shc but not FAK seems to mediate this effect. These results show a critical role of PTEN in metastasis of RCC, depending on protein phosphatase activity via Shc. This new insight opens an alley of additional approaches complementing current cancer therapy and metastasis prediction in RCC.

The TLR7 agonist R848 alleviates allergic inflammation by targeting invariant NKT cells to produce IFN-gamma.

It has been documented that TLR7 stimulation triggers not only antiviral responses, but also alleviates experimental asthma. Considering the implication of invariant NKT (iNKT) cells in both situations, we postulated that they might contribute to the anti-inflammatory effect of TLR7 ligands. We show in this study that spleen cells activated by the TLR7 agonist resiquimod (R848) attenuate allergic inflammation upon adoptive transfer when they are recovered from wild-type, but not from iNKT cell-deficient Jα18(-/-) mice, which proves the specific involvement of this regulatory population. Furthermore, we provide evidence that IFN-γ is critical for the protective effect, which is lost when transferred iNKT cells are sorted from IFN-γ-deficient mice. In support of a direct activation of iNKT cells through TLR7 signaling in vivo, we observed a prompt increase of serum IFN-γ levels, associated with upregulation of CD69 expression on iNKT cells. Moreover, we demonstrate that iNKT cells effectively express TLR7 and respond to R848 in vitro by producing high levels of IFN-γ in the presence of IL-12, consistent with the conclusion that their contribution to the alleviation of allergic inflammation upon treatment with TLR7 ligands is mediated through IFN-γ.

Basophils: new players in the cytokine network.

Basophils belong to a myeloid cell population that has been ignored for more than a century, mainly because of its paucity, its lack of specific markers, and the absence of experimental models. Given that in mice, even the mere existence of basophils was contested, they were alluded to as "histamine-producing cells" or "non-T non-B cells" in initial studies. It is now widely acknowledged that basophils respond to various IgE-dependent or -independent stimuli, and are engaged in a complex cross talk with a number of immunocompetent cells (T or B lymphocytes, macrophages, dendritic cells, endothelial cells…). Indeed, on the one hand they are critically involved during the onset, the effector phase and exacerbation of T(H)2 responses through their capacity to generate large amounts of cytokines with pro-T(H)2 functions (IL-4, IL-13 TSLP, IL-25), on the other hand, they contribute to immunoglobulin synthesis and class switching, angiogenesis, autoimmunity, tumor immunity and hematopoiesis by producing cytokines such as IL-6, VEGF, GM-CSF and IL-3. Finally, it has been established that they can present antigens to CD4+ or CD8+ T cells in an MHC class II- or class I-dependent manner, respectively. Taken together, these activities confer important immunoregulatory functions upon basophils, both in innate and adaptive immunity.

Adhesion of renal carcinoma cells to endothelial cells depends on PKCmu.

BACKGROUND: The formation of metastases includes the separation of tumor cells from the primary tumor, cell migration into subendothelial tissue and cell proliferation in secondary organ. In this process, cell adhesion of tumor cells to the endothelium is an essential requirement for formation of metastases. Protein kinase C (PKC) regulates adhesion and proliferation. To identify a relation between PKC isoforms and tumor progression in renal cell carcinoma (RCC), the influence of PKC isoforms on cell adhesion and proliferation, and possible influences of integrins were analyzed in RCC cells. METHODS: The experiments were performed in the RCC cell lines CCF-RC1 and CCF-RC2 after pre-incubation (16 h) with the PKC inhibitors GF109203X (inhibits PKCalpha, betaI, betaII, gamma, delta and epsilon), GO6976 (inhibits PKCalpha, betaI and mu), RO31-8220 (inhibits PKCalpha, betaI, betaII, gamma and epsilon) and rottlerin (inhibits PKCdelta). Cell adhesion was assessed through adherence of RCC cells to an endothelial monolayer. Cell proliferation was analyzed by a BrdU incorporation assay. The expression of beta1 integrins was analyzed by flow cytometry. RESULTS: In CCF-RC1 cells, cell adhesion was significantly reduced by GO6976 to 55% and by RO31-8220 to 45% of control. In CCF-RC2 cells, only GO6976 induced a significant reduction of cell adhesion to 50% of control levels. Proliferation of both cell lines was reduced by rottlerin to 39% and 45% of control, respectively. The beta1 integrin expression on the cell surface of CCF-RC1 and CCR-RC2 cells was decreased by RO31-8220 to 8% and 7% of control, respectively. beta2 and beta3 integrins were undetectable in both cell lines. CONCLUSIONS: The combination of the PKC inhibitors leads to the assumption that PKCmu influences cell adhesion in CCF-RC1 and CCF-RC2 cells, whereas in CCF-RC1 cells PKCepsilon also seems to be involved in this process. The expression of beta1 integrins appears to be regulated in particular by PKCepsilon. Cell proliferation was inhibited by rottlerin, so that PKCdelta might be involved in cell proliferation in these cells.

Histamine, immune cells and autoimmunity.

Histamine is one ofthe most versatile biogenic amines with multiple roles during the immune response and in allergic disorders. With four distinct G protein-coupled receptors (H1R, HER, H3R and H4R), intracellular histamine binding sites (most likely members of the cytochrome P450 family) as well as a membrane transporter (Organic Cation Transporter; OCT3) expressed in various immunocompetent cells, it can entertain a complex network of interactions. These signaling pathways are expressed differentially, depending on the stage of differentiation or activation of target cells, thus adding a further degree of complexity to the system. For this reason, published data are sometimes conflicting and varying according to the particular cell type or responses analyzed and the experimental approaches used. On the other hand, histamine is generated by several cells during the immune response, not only through release of intracellular stores in mast cells or basophils in response to IgE-dependent or -independent stimuli, but also through neosynthesis catalyzed by histidine decarboxylase (HDC) in a number ofhematopoietic cells that secrete the amine immediately without prior storage. These features enable histamine to tune the fine balance between immunity and tolerance by affecting dendritic cells, immunoregulatory cells, T-cell polarization and cytokine production, making the way for new pharmacological strategies to control immune reactivity during immune disorders, such as autoimmunity.

IL-33 activates unprimed murine basophils directly in vitro and induces their in vivo expansion indirectly by promoting hematopoietic growth factor production.

IL-33, a new member of the IL-1 family, has been described as an important inducer of Th2 cytokines and mediator of inflammatory responses. In this study, we demonstrate that murine basophils sorted directly from the bone marrow, without prior exposure to IL-3 or Fc(epsilon)R cross-linking, respond to IL-33 alone by producing substantial amounts of histamine, IL-4, and IL-6. These cells express ST2 constitutively and generate a cytokine profile that differs from their IL-3-induced counterpart by a preferential production of IL-6. In vivo, IL-33 promotes basophil expansion in the bone marrow (BM) through an indirect mechanism of action depending on signaling through the beta(c) chain shared by receptors for IL-3, GM-CSF, and IL-5. IL-3 can still signal through its specific beta(IL-3) chain in these mutant mice, which implies that it is not the unique growth-promoting mediator in this setup, but requires IL-5 and/or GMCSF. Our results support a major role of the latter growth factor, which is readily generated by total BM cells as well as sorted basophils in response to IL-33 along with low amounts of IL-3. Furthermore, GM-CSF amplifies IL-3-induced differentiation of basophils from BM cells, whereas IL-5 that is also generated in vivo, affects neither their functions nor their growth in vitro or in vivo. In conclusion, our data provide the first evidence that IL-33 not only activates unprimed basophils directly, but also promotes their expansion in vivo through induction of GM-CSF and IL-3.

H4 histamine receptors mediate cell cycle arrest in growth factor-induced murine and human hematopoietic progenitor cells.

The most recently characterized H4 histamine receptor (H4R) is expressed preferentially in the bone marrow, raising the question of its role during hematopoiesis. Here we show that both murine and human progenitor cell populations express this receptor subtype on transcriptional and protein levels and respond to its agonists by reduced growth factor-induced cell cycle progression that leads to decreased myeloid, erythroid and lymphoid colony formation. H4R activation prevents the induction of cell cycle genes through a cAMP/PKA-dependent pathway that is not associated with apoptosis. It is mediated specifically through H4R signaling since gene silencing or treatment with selective antagonists restores normal cell cycle progression. The arrest of growth factor-induced G1/S transition protects murine and human progenitor cells from the toxicity of the cell cycle-dependent anticancer drug Ara-C in vitro and reduces aplasia in a murine model of chemotherapy. This first evidence for functional H4R expression in hematopoietic progenitors opens new therapeutic perspectives for alleviating hematotoxic side effects of antineoplastic drugs.

The pro-Th2 cytokine IL-33 directly interacts with invariant NKT and NK cells to induce IFN-gamma production.

IL-33 has recently been identified as a cytokine endowed with pro-Th2 functions, raising the question of its effect on invariant natural killer T cell (iNKT), which are potent IL-4 producers. Here, we report a two-fold increase of iNKT-cell counts in spleen and liver after a 7-day treatment of mice with IL-33, which results from a direct effect, given that purified iNKT cells express the T1/ST2 receptor constitutively and respond to IL-33 by in vitro expansion and functional activation. Conversely to the expected pro-Th2 effect, IL-33 induced a preferential increase in IFN-gamma rather than IL-4 production upon TCR engagement that depended on endogenous IL-12. Moreover, in combination with the pro-inflammatory cytokine IL-12, IL-33 enhanced IFN-gamma production by both iNKT and NK cells. Taken together these data support the conclusion that IL-33 can contribute as a co-stimulatory factor to innate cellular immune responses.

Cutting edge: histamine receptor H4 activation positively regulates in vivo IL-4 and IFN-gamma production by invariant NKT cells.

Histamine (HA) is a biogenic amine with multiple activities in the immune system. In this study we demonstrate that histamine-free histidine decarboxylase-deficient (HDC(-/-)) mice present a numerical and functional deficit in invariant NK T (iNKT) cells as evidenced by a drastic decrease of IL-4 and IFN-gamma production. This deficiency was established both by measuring cytokine levels in the serum and intracellularly among gated iNKT cells. It resulted from the lack of HA, because a single injection of this amine into HDC(-/-) mice sufficed to restore normal IL-4 and IFN-gamma production. HA-induced functional recovery was mediated mainly through the H4 histamine receptor (H4R), as assessed by its abrogation after a single injection of a selective H4R antagonist and the demonstration of a similar iNKT cell deficit in H4R(-/-) mice. Our findings identify a novel function of HA through its H4R and suggest that it might become instrumental in modulating iNKT cell functions.