Hagen, P.M. van

Erratum to "High-dose intravenous immunoglobulin does not reduce the numbers of circulating CD14+CD16++ monocytes in patients with inflammatory disorders" [Clin. Immunol. 145 (2012) 11-12] (Article)

2013-04-01T00:00:00Z

Combined TCRG and TCRA TREC analysis reveals increased peripheral T-lymphocyte but constant intra-thymic proliferative history upon ageing (Article)

2013-03-01T00:00:00Z

T-cell receptor (TCR) repertoire diversity, thymic output, clonal size and peripheral T-lymphocyte numbers largely depend on intra-thymic and post-thymic T-lymphocyte proliferation. However, quantitative insight into thymocyte and T-lymphocyte proliferation is still lacking. We developed a new TCRG-based TCR excision circle (TREC) assay, the Vγ-Jγ TREC assay, which we used together with an adjusted δREC-ψJα TREC assay to quantify the proliferative history of human thymocyte and T-lymphocyte subpopulations from children and adults. This revealed that thymocytes undergo ∼6-8 intra-thymic cell divisions from the double negative (DN) 3 developmental stage onwards, which appeared independent of age. Thus thymocyte proliferation after the DN3 developmental stages is stable and therefore not contributing to the reduced thymic output upon ageing. Cord blood naive T lymphocytes had already undergone ∼2-3 post-thymic cell divisions, which increased to ∼6-7 cell divisions in naive T lymphocytes of middle-aged adults, indicating the importance of homeostatic naive T-lymphocyte proliferation from a young age onwards in the maintenance of peripheral T-lymphocyte numbers. In conclusion, our data provide quantitative insight into the proliferative history of thymocyte and T-lymphocyte subpopulations and alterations herein upon ageing. This novel TREC assay approach could prove valuable in immune status monitoring in a variety of conditions.

Perigranuloma localization and abnormal maturation of B cells emerging key players in sarcoidosis? (Article)

2013-02-15T00:00:00Z

Rationale: Recent observations of abnormal immunoglobulin responses and case reports describing successful B-cell ablative therapy suggest involvement ofBcells in the pathogenesis of sarcoidosis. Objectives: To investigate how abnormal B-cell maturation and function in patients with sarcoidosis contribute to disease. Methods: Patients with sarcoidosis (n = 32) were included for detailed analysis by immunohistochemistry of tissue, flow cytometry of blood B-cell subsets, andserumimmunoglobulin levels.Vaccinationresponses in patients with sarcoidosis to influenza virus and encapsulated bacteria andmolecular analysis ofimmunoglobulin heavy chain transcriptswere studied for functional analysis of immunoglobulin responses. Measurements and Main Results: Perigranuloma localization of IgAproducing plasma cells and numerous B cells were found in affected tissues. Total blood B-cell numbers were normal, CD271 memory B cells were significantly reduced, and CD272IgA1 B cells were significantly increased; the results are normalized in patients treated with TNF-ablockers.Despitethis,patientshadnormal serumimmunoglobulin levels and normal antigen-specific immunoglobulin responses. IgAand IgGtranscripts, however, showed highfrequencies of somatic hypermutations and increased usage of downstream IgG subclasses, suggestive for prolonged or repetitive responses. Conclusions: The large B-cell infiltrates in granulomatous tissue and increased molecular signs of antibody maturation are indicative of direct involvement of B cells in local inflammatory processes in patients with sarcoidosis. Moreover, CD272IgA1 B cells could be a marker for treatment with TNF-a blockers. These findings of B cells as emerging key players provide a rationale for a systematic study on B-cell ablative therapy in patients with sarcoidosis. Copyright

Morbidly obese human subjects have increased peripheral blood CD4 + T cells with skewing toward a Treg- and Th2-dominated phenotype (Article)

2012-02-01T00:00:00Z

Obesity is associated with local T-cell abnormalities in adipose tissue. Systemic obesity-related abnormalities in the peripheral blood T-cell compartment are not well defined. In this study, we investigated the peripheral blood T-cell compartment of morbidly obese and lean subjects. We determined all major T-cell sub-populations via six-color flow cytometry, including CD8+and CD4+T cells, CD4+T-helper (Th) subpopulations, and natural CD4+CD25+FoxP3+T-regulatory (Treg) cells. Moreover, molecular analyses to assess thymic output, T-cell proliferation (T-cell receptor excision circle analysis), and T-cell receptor-β (TCRB) repertoire (GeneScan analysis) were performed. In addition, we determined plasma levels of proinflammatory cytokines and cytokines associated with Th subpopulations and T-cell proliferation. Morbidly obese subjects had a selective increase in peripheral blood CD4+naive, memory, natural CD4+CD25+FoxP3+Treg, and Th2 T cells, whereas CD8+T cells were normal. CD4+and CD8+T-cell proliferation was increased, whereas the TCRB repertoire was not significantly altered. Plasma levels of cytokines CCL5 and IL-7 were elevated. CD4+T-cell numbers correlated positively with fasting insulin levels. The peripheral blood T-cell compartment of morbidly obese subjects is characterized by increased homeostatic T-cell proliferation to which cytokines IL-7 and CCL5, among others, might contribute. This is associated with increased CD4+T cells, with skewing toward a Treg- and Th2-dominated phenotype, suggesting a more anti-inflammatory set point.

B-cell replication history and somatic hypermutation status identify distinct pathophysiologic backgrounds in common variable immunodeficiency (Article)

2011-12-22T00:00:00Z

Common variable immunodeficiency disorder (CVID) is the most prevalent form of primary idiopathic hypogammaglobulinemia. Identification of genetic defects in CVID is hampered by clinical and immunologic heterogeneity. By flow cytometric immunophenotyping and cell sorting of peripheral B-cell subsets of 37 CVID patients, we studied the B-cell compartment at the B-cell subset level using the κ-deleting recombination excision circle assay to determine the replication history and the Igκ-restriction enzyme hot-spot mutation assay to assess the somatic hypermutation status. Using this approach, 5 B-cell patterns were identified, which delineated groups with unique replication and somatic hypermutation characteristics. Each B-cell pattern reflected an immunologically homogenous patient group for which we proposed a different pathophysiology: (1) a B-cell production defect (n ∇ 8, 18%), (2) an early peripheral B-cell maturation or survival defect (n ∇ 4, 11%), (3) a B-cell activation and proliferation defect (n ∇ 12, 32%), (4) a germinal center defect (n ∇ 7, 19%), and (5) a postgerminal center defect (n ∇ 6, 16%). The results of the present study provide for the first time insight into the underlying pathophysiologic background in 5 immunologically homogenous groups of CVID patients. Moreover, this study forms the basis for larger cohort studies with the defined homogenous patient groups and will facilitate the identification of underlying genetic defects in CVID.

The neonatal Fc receptor is expressed by human retinal pigment epithelial cells and is downregulated by tumour necrosis factor-alpha (Article)

2011-06-01T00:00:00Z

Background/aims: The neonatal Fc receptor (FcRn) protects immunoglobulin G (IgG) from catabolism, controls its transport between cell layers and extends its serum half-life. In the human, vitreous IgG can be found, but how vitreous IgG is processed or transported is currently unknown. The FcRn is a candidate molecule to regulate these processes. The authors examined FcRn expression and regulation in human retinal pigment epithelium (RPE) cells. Methods: In three primary RPE cell cultures (from three donor eyes) and in the human RPE cell line ARPE-19, FcRn and beta-2-microglobulin (β2M) mRNA levels were determined by real-time quantitative PCR. FcRn protein expression was analysed by western blot studies. Stimulation experiments were performed with recombinant human tumour necrosis factor (TNF)-α and interferon (IFN)-γ. HT-29, THP-1 and HeLa cell lines were used as FcRn positive and negative non-ocular controls, respectively. Results: Expression of FcRn mRNA and protein was demonstrated in all three RPE cultures. After stimulation with TNF-α, FcRn expression is downregulated in RPE cells and upregulated in HT-29 and THP-1 cells. IFN-γ has no effect on FcRn expression in RPE cells. Conclusions: Human RPE cells express FcRn. The proinflammatory cytokine TNF-α downregulates FcRn expression. The authors speculate that the FcRn may play a pivotal role in the immune privilege of the human eye.

Whole orbital tissue culture identifies imatinib mesylate and adalimumab as potential therapeutics for Graves' ophthalmopathy (Article)

2011-05-01T00:00:00Z

Background and aims: Biologicals and small inhibitory molecules are used to treat inflammatory diseases, but their efficacy varies upon clinical application. Using a whole orbital tissue culture system, we tested the potential efficacy of imatinib mesylate (a tyrosine kinase inhibitor that blocks platelet-derived growth factor (PDGF)-receptor, c-Abl and c-Kit activity) and adalimumab (an anti-TNF-α antibody) for the treatment of Graves' ophthalmopathy (GO). Methods: Orbital fat tissue from GO patients (n=10) was cultured with or without imatinib mesylate or adalimumab. PDGF-B and tumour necrosis factor (TNF)-α mRNA expression levels were determined in the primary orbital tissue, and interleukin (IL)-6 and hyaluronan were measured in tissue-culture supernatants. Results: Imatinib mesylate significantly (p=0.005) reduced IL-6 and hyaluronan production. The inhibition of hyaluronan production correlated positively and significantly (p<0.05) with the PDGF-B mRNA level in the primary tissue. Adalimumab also significantly (p=0.005) reduced IL-6 production. The amount of IL-6 inhibition correlated positively with the TNF-α mRNA level in the primary tissue, but this was not significant. Conclusions: Imatinib mesylate can be expected to reduce inflammation and tissue remodelling in GO, while adalimumab can be mainly expected to reduce inflammation. This in vitro tissue-culture model may, in future, prove valuable to test novel therapeutics for their presumed effect in GO as well as in other inflammatory diseases.

Use of TNF blockers and other targeted therapies in rare refractory immune-mediated inflammatory diseases: Evidence-based or rational? (Article)

2010-12-01T00:00:00Z

Evidence-based medicine implies that clinical decision making should be based on external research evidence when available. This external evidence includes, but is certainly not restricted to, randomised controlled trials (RCTs). The development of powerful but often expensive targeted therapies for immune-mediated inflammatory diseases (IMIDs) is one of the major successes of evidence-based medicine but, paradoxically, also threatens the traditional RCT-based approach. Indeed, the increasing availability of these drugs decreases the number of patients available for RCTs, questions the ethical basis for the use of placebo groups and raises the issue of cost-efficacy. These considerations become even more important in rare phenotypically diverse and potentially life- or organ-threatening IMIDs such as sarcoidosis, Behçet's disease and uveitis. Using the successful application of tumour necrosis factor blockade in these diseases as an example, this review defends the concept that pathophysiological insights in cellular and molecular disease pathways as well as limited case series are valid sources of external evidence for the rational use of targeted therapies in these rare refractory conditions. If authors fail to redefine their concept of rational therapy along the lines of not only evidence-based but also pathophysiology-based and practice-based medicine, they may underestimate the potential of novel drugs in rare refractory IMIDs and thereby jeopardise the health of their patients.

Platelet-derived growth factor-BB: a stimulus for cytokine production by orbital fibroblasts in Graves' ophthalmopathy. (Article)

2010-01-01T00:00:00Z

PURPOSE: Graves' ophthalmopathy (GO) is characterized by the infiltration of immune cells into the orbit, a process in which cytokines play a central role. Orbital fibroblasts are potent producers of cytokines on different stimuli. Recently, the authors showed increased expression of the PDGF-B chain in GO orbital tissue. The dimeric PDGF-BB molecule has been described to activate the NF-kappaB pathway, which is well recognized for its role in regulating cytokine production. This study was conducted to determine the role of PDGF-BB in the production of proinflammatory cytokines by orbital fibroblasts in GO. METHODS: Orbital, lung, and skin fibroblasts were stimulated with PDGF-BB, and cytokine (IL-1beta, IL-6, IL-8, IL-16, CCL2, CCL5, CCL7, TNF-alpha) production was measured by ELISA. Involvement of NF-kappaB activation through PDGF signaling was investigated by electrophoretic mobility shift assay, specific NF-kappaB inhibitors, and the PDGF-receptor kinase inhibitor imatinib mesylate. RESULTS: IL-6, IL-8, CCL2, CCL5, and CCL7 production by orbital fibroblasts was increased by PDGF-BB stimulation, whereas IL-16, IL-1beta, and TNF-alpha production was not affected. PDGF-BB induced NF-kappaB activity in orbital fibroblasts, and both NF-kappaB inhibitors and imatinib mesylate reduced PDGF-BB-induced cytokine production. Similar, but less vigorous, effects of PDGF-BB on cytokine production were observed in lung and skin fibroblasts. CONCLUSIONS: PDGF-BB is a potent inducer of proinflammatory cytokines via the NF-kappaB pathway in orbital fibroblasts, whereas cytokine production by fibroblasts from other anatomic locations showed a moderate response. These data suggest a possible role for PDGF-BB in regulating orbital inflammation in GO and identify the PDGF signaling cascade as a therapeutic target in GO.

Imatinib mesylate and AMN107 inhibit PDGF-signaling in orbital fibroblasts: A potential treatment for Graves' ophthalmopathy (Article)

2009-12-01T00:00:00Z

PURPOSE. Excessive orbital fibroblast proliferation and hyaluronan production are characteristic of Graves' ophthalmopathy (GO) and are driven by local mediators. Imatinib mesylate and AMN107 are tyrosine kinase inhibitors that inhibit fibroblast proliferation and collagen production in lungs and skin. This study was conducted to determine whether imatinib mesylate and AMN107 inhibit orbital fibroblast proliferation and hyaluronan production induced by PDGF-BB and TGF-β1and whether expression of the genes PDGF-B and TGF-B1(growth factors suggested to play a role in GO) are increased in GO orbital tissues. METHODS. PDGF-B and TGF-B1mRNA levels were determined in orbital tissues of 13 patients with GO and 5 control patients. Orbital fibroblasts were cultured from eight patients with GO and three control patients and the effect of imatinib mesylate and AMN107 on PDGF-BB and TGF-β1-induced orbital fibroblast proliferation, signaling cascades, hyaluronan synthase (HAS) gene expression and hyaluronan production were determined. RESULTS. PDGF-B and TGF-B1mRNA levels were significantly increased in GO orbital tissues. Imatinib mesylate and AMN107 inhibited PDGF-BB-induced orbital fibroblast proliferation, HAS induction and hyaluronan production by blocking PDGFreceptor phosphorylation. TGF-β1induced HAS expression and hyaluronan production. This induction was not inhibited by imatinib mesylate or AMN107, due to the inability of TGF-β1to activate c-Abl kinase activity in orbital fibroblasts. CONCLUSIONS. Imatinib mesylate and AMN107 inhibit orbital fibroblast proliferation and hyaluronan production induced by PDGF-BB; a factor highly expressed in orbital tissue from patients with GO. The drugs, however, had no effect on TGF-β1-induced HAS expression and hyaluronan production. Nevertheless, imatinib mesylate and AMN107 should be considered as treatment candidates for GO.

Complement factor H Y402H decreases cardiovascular disease risk in patients with familial hypercholesterolaemia (Article)

2009-06-24T00:00:00Z

AimsActivation of the complement system seems an important link between inflammation and atherogenesis. The Y402H polymorphism of complement factor H (CFH) has been associated with cardiovascular events, but results are conflicting and possibly modified by age of onset of cardiovascular disease (CVD).Methods and resultsWe determined whether or not the Y402H polymorphism influenced CVD risk in a multicentre cohort study involving 2016 unrelated patients with familial hypercholesterolaemia (FH), who have an extremely increased susceptibility to premature CVD. We identified 261 individuals who were homozygous for the polymorphism (CC genotype; 12.9), 929 individuals who were heterozygous (TC genotype; 46.1), and 826 individuals carried the wild-type (TT genotype; 41.0). During 95 115 person years, 644 patients had a cardiovascular event. Carriers of the CC genotype had a decreased risk of CVD (hazard ratio 0.67, 95 confidence interval 0.51-0.87; P = 0.003) relative to the other genotype groups. This association was unaltered after adjustment for clinically relevant cardiovascular risk factors or age effects.ConclusionAmong patients with severely increased risk of early onset CVD, the Y402H CFH variant was inversely associated with susceptibility to CVD. This suggests that CFH is a modifier gene of CVD.

Do synovial biopsies help to support evidence for involvement of innate immunity in the immunopathology of Behçet's disease? (Article)

2009-04-30T00:00:00Z

Behçet's disease is a complex vasculitis of unknown etiology. Abundant neutrophils suggest the involvement of innate immunity. Cytokines are skewed to the T-helper-1 pattern. Few sterile organs are easily accessible for analysis in Behçet's disease. Cañete and coworkers identify inflamed joints as a feasible model and suggest the involvement of innate immunity in Behçet's disease.

Immunophenotyping of mast cells: A sensitive and specific diagnostic tool for systemic mastocytosis (Article)

2009-01-01T00:00:00Z

Introduction: The diagnosis of systemic mastocytosis (SM) is based on a combination of major and minor criteria. Flow cytometric detection of aberrant expression of CD2 and/or CD25 on CD117-positive mast cells is one of the minor criteria used. In the present study we examined the sensitivity and specificity of mast cell immunophenotyping in the diagnosis of SM. Material and methods: Patients were 36 persons with systemic mastocytosis diagnosed according to WHO criteria. Controls were 31 patients without SM. Immunophenotyping was performed according to published guidelines. Results: All patients with SM were positive for CD2 and/or CD25. All patients without SM, except one, were negative for these markers. The sensitivity for immunophenotyping was 100%, the specificity 91%. The positive and negative predictive values were 97% and I00% respectively. Conclusion: Immunophenotyping of bone marrow derived mast cells is not only a very sensitive but also a very specific method to diagnose SM with high positive and negative predictive value.

Mycophenolate sodium: Effective treatment for therapy-refractory intestinal Behçet's disease, evaluated with enteroscopy (Article)

2008-12-01T00:00:00Z

Is imatinib mesylate a promising drug in systemic sclerosis? (Article)

2008-08-01T00:00:00Z

A patient with therapy-resistant and progressive systemic sclerosis (SSc) with pulmonary involvement who was treated with imatinib mesylate is described herein. Prior to treatment, pulmonary fibroblasts obtained from the patient were cultured and incubated with imatinib mesylate. Preincubation of the fibroblasts for 16 hours with 2.5 μg/ml imatinib mesylate efficiently abrogated platelet-derived growth factor BB-induced fibroblast proliferation. Furthermore, transforming growth factor β1-induced type I collagen gene transcription was blocked. During treatment, the patient's pulmonary involvement stabilized and her skin tightness improved. To our knowledge, this is the first report of a patient with therapy-refractory SSc responding to treatment with imatinib mesylate.

The role of cortistatin in the human immune system (Article)

2008-05-14T00:00:00Z

Cortistatin (CST) is a recently described neuropeptide that shares high homology with somatostatin (somatotropin release-inhibiting factor, SRIF) and binds with high affinity to all somatostatin (sst) receptor subtypes. CST is currently known to have a widespread distribution in many human organs including the immune system. The activities specific to CST may be partially attributable to its binding to the growth hormone secretagogue (GHS)-receptor (GHS-R) and the orphan G-protein-coupled receptor MrgX2. Human immune cells produce CST, whereas macrophage lineage and activated endothelium express sst2, and human lymphocytes express sst3. The human thymus expresses sst1, 2, 3, MrgX2 and almost all immune cells express GHS-R. Moreover, at this very moment promising research with CST in experimental animal models is being performed. On the basis of these promising results, studies aiming to further evaluate the possibilities of CST as a therapeutic agent in human immune-mediated inflammatory diseases are warranted.

The EUROclass trial: Defining subgroups in common variable immunodeficiency (Article)

2008-01-01T00:00:00Z

The heterogeneity of common variable immunodeficiency (CVID) calls for a classification addressing pathogenic mechanisms as well as clinical relevance. This European multicenter trial was initiated to develop a consensus of 2 existing classification schemes based on flowcytometric B-cell phenotyping and the clinical course. The clinical evaluation of 303 patients with the established diagnosis of CVID demonstrated a significant coincidence of granulomatous disease, autoimmune cytopenia, and splenomegaly. Phenotyping of B-cell subpopulations confirmed a severe reduction of switched memory B cells in most of the patients that was associated with a higher risk for splenomegaly and granulomatous disease. An expansion of CD21lowB cells marked patients with splenomegaly. Lymphadenopathy was significantly linked with transitional B-cell expansion. Based on these findings and pathogenic consideration of B-cell differentiation, we suggest an improved classification for CVID (EUROclass), separating patients with nearly absent B cells (less than 1%), severely reduced switched memory B cells (less than 2%), and expansion of transitional (more than 9%) or CD21lowB cells (more than 10%). Whereas the first group contains all patients with severe defects of early B-cell differentiation, severely reduced switched memory B cells indicate a defective germinal center development as found in inducible constimulator (ICOS) or CD40L deficiency. The underlying defects of expanded transitional or CD21lowB cells remain to be elucidated. This trial is re-gistered at http://www.uniklinik-freiburg.de/zks/live/uklregister/Oeffentlich.html as UKF000308.

Octreotide Long-Acting Repeatable for the Treatment of Chronic Macular Edema in Uveitis (Article)

2007-12-01T00:00:00Z

Purpose: To report on the efficacy of the somatostatin analog octreotide long-acting repeatable (LAR), in the treatment of uveitic chronic macular edema (CME). Design: Case series, retrospective analysis. Methods: In 20 patients, 20 episodes of recurrent CME during otherwise quiescent uveitis were treated with intramuscular octreotide LAR injections. Patients were included if CME control with acetazolamide or systemic and periocular steroids had failed during previous CME episodes or if contraindications existed for persistent use of these therapies. Mean outcome points were CME and visual acuity changes. Correlation of prognostic factors with these outcomes was analyzed. Results: The included CME episodes occurred 7.6 ± 1.4 years after onset of uveitis. Octreotide LAR treatment started 7.0 ± 7.3 months after diagnosis of CME. CME decreased in 70% of episodes, after 2.7 ± 1.3 months of treatment. After arrest of successful treatment, CME recurred instantly (27.2%) or within six months (36.4%). In 36.4% of successfully treated episodes, CME was absent for more than one year. A probable prognostic factor for success was the duration of CME before treatment. Conclusions: Octreotide LAR had an edema-reducing effect in 70% of treated CME episodes. Successful response was related to duration of CME before start of treatment. The early recurrence of CME (63.6%) after arrest of octreotide LAR advocates a long-term treatment in recent episodes of macular edema in otherwise quiescent uveitis.

Adalimumab: A new modality for Behçet's disease? [7] (Article)

2007-04-01T00:00:00Z

Anti-TNF-alpha therapy for sight threatening uveitis. (Article)

2005-05-01T00:00:00Z

AIM: To describe the effect of additional treatment with anti-TNF-alpha therapy in a case series of 13 patients with serious sight threatening uveitis. METHODS: 13 patients with serious sight threatening uveitis were included, of whom six had Behcet's disease, five had idiopathic posterior uveitis, one had sarcoidosis, and one birdshot retinochoroiditis. Onset and course of ocular inflammation, inflammatory signs, and visual acuity were assessed. Patients were treated with 200 mg (approximately 3 mg/kg) infliximab infusion. Repeat infusions were given based on clinical response. RESULTS: Infliximab treatment resulted in an effective suppression of ocular inflammation in all patients. In patients with non-Behcet's disease uveitis visual acuity in six out of eight improved or was stable. In patients with Behcet's disease visual acuity in five out of six improved or was stable. CONCLUSION: Anti-TNF-alpha treatment may be of value in the treatment of uveitis, and in patients with Behcet's disease, leading to suppression of ocular inflammation, vasculitis, and improvement of vision in the majority. Based on these results a controlled masked study is warranted.

Autologous peripheral retinal pigment epithelium translocation in patients with subfoveal neovascular membranes (Article)

2004-01-01T00:00:00Z

AIM: To evaluate the possibility of translocating autologous peripheral retinal pigment epithelial (RPE) cells and enhance their adhesion to improve functional outcome after choroidal neovascular membrane extraction in patients with subfoveal neovascular membranes. METHODS: A prospective, non-controlled surgical study in eight consecutive patients operated between February and July 2001 with final data monitoring in July 2002. All patients had mixed subfoveal membranes of 2-4 disc diameters. Functional tests included Snellen vision and central fixation testing. During vitrectomy, after the extraction of the neovascular complex, 8 x 10(4)-16 x 10(4) RPE cells were removed from the periphery and translocated under the macula following the submacular injection of 2 microg of poly-L-lysine to promote adhesion of the cells. RESULTS: With a follow up ranging from 3 months to 16 months, a pigmented area was seen in the extraction bed of the neovascular membrane in only one patient. Fixation was at the edge of the extraction bed in three patients. Vision remained the same in five patients and deteriorated in three (all with retinal detachment). Retinal detachment due to proliferative vitreoretinopathy occurred in three patients. CONCLUSIONS: The translocation of autologous peripheral RPE cells after membrane extraction was technically possible in a sterile manner, but was associated with a high proliferative vitreoretinopathy rate and in the present series had no measurable positive effect on functional outcome.

Differential expression of somatostatin receptor subtypes in human peripheral blood mononuclear cell subsets (Article)

2004-01-01T00:00:00Z

BACKGROUND: Somatostatin (SS)-binding sites have been demonstrated in human lymphoid tissues and peripheral blood cells. However, not much is known with respect to the SS receptor subtype (sst) expression pattern and the expression of SS itself in the immune system. OBJECTIVE: The aim of this study was to evaluate the mRNA expression of the five known sst (sst(1-5)) in peripheral blood mononuclear cell (sub)populations. Moreover, the expression of the mRNAs encoding SS and the SS-like peptide cortistatin (CST) in immune cell subsets was studied. METHODS: RT-PCR and quantitative PCR were performed to evaluate sst, SS and CST mRNA expression in cells in the basal or activated state. Fluorescence-activated cell sorter (FACS) analysis using fluorescent SS was performed to visualize sst protein on cell membranes. RESULTS: B- and T-lymphocytes selectively expressed sst(3) mRNA. sst(3) expression in B-lymphocytes was significantly lower compared with T-lymphocytes. Unstimulated, freshly isolated monocytes did not express any sst mRNA. Upon activation, monocytes selectively expressed sst(2) mRNA, whereas T-lymphocyte activation upregulated sst(3) expression. sst(2) mRNA expression on monocytes was confirmed by FACS analysis. B- and T-lymphocytes did not express SS mRNA, while both cell types expressed CST mRNA. CST mRNA expression was downregulated following T-lymphocyte activation. CONCLUSION: We demonstrate for the first time unequivocally that human peripheral blood B- and T-lymphocytes selectively express sst(3), whereas monocytes do not express sst. However, upon activation, monocytes are induced to express sst(2A). No expression of SS mRNA was detected in any cell type, whereas all cell types expressed CST mRNA. The differential expression of sst and CST mRNA in lymphocytes and monocytes suggests a functional significance for the CST-sst interaction in immune cells, but further studies should be performed to evaluate the significance of sst and CST in these cells.

Cortistatin rather than somatostatin as a potential endogenous ligand for somatostatin receptors in the human immune system (Article)

2003-01-01T00:00:00Z

Cells of the human immune system have been shown to express somatostatin receptors (sst). The expression of sst suggests a functional role of the peptide somatostatin (SS). However, SS expression has not been demonstrated yet in different human immune tissues. Therefore, we investigated by RT-PCR the expression of both SS and cortistatin (CST), a SS-like peptide, in various human lymphoid tissues and immune cells. We detected SS mRNA expression in the human thymus only, while not in thymocytes. CST mRNA was clearly expressed in the immune cells, lymphoid tissues, and bone marrow. Using quantitative RT-PCR, significant differences in expression levels between tissues were demonstrated. Expression of CST mRNA was up-regulated during differentiation of monocytes into macrophages and dendritic cells and could be up-regulated by lipopolysaccharide stimulation. Two differently sized cDNA fragments of CST were detected in the majority of cells and tissues. However, although both fragments were detected in nearly all T-cell lines (7 of 8), most of the B-cell lines expressed the short fragment only (8 of 10). Using autoradiography, we showed that CST displaced [125I-Tyr3]octreotide binding with relatively high affinity on human thymic tissue and sst2-expressing cells. This is the first extensive study demonstrating that human lymphoid tissues and immune cells express different levels of CST mRNA and that its expression can be regulated. On the basis of these observations, we hypothesize a role for CST as an endogenous ligand of at least the sst2 receptor in the human immune system, rather than SS itself.

Expression of somatostatin, cortistatin, and somatostatin receptors in human monocytes, macrophages, and dendritic cells. (Article)

2003-01-01T00:00:00Z

Increasing evidence suggests that neuropeptides play a role in the regulatory mechanisms between the neuroendocrine and immune systems. A differential expression of the five known somatostatin (SS) receptors (sst1-5) has been demonstrated in human immune cells and tissues. However, little is known concerning regulation and expression of sst1-5 and the peptide SS. Therefore, we investigated the expression and the time-dependent regulation of sst1-5, SS, and cortistatin (CST), a novel SS-like peptide, in human monocytes (MO), monocyte-derived macrophages (MP), and dendritic cells (DC) in the basal and lipopolysaccharide (LPS)-activated state. MO, MP, and DC selectively expressed sst2 mRNA. SS mRNA was not detectable, whereas all samples expressed CST mRNA. Expression levels of sst2 and CST mRNA showed marked differences and were in the rank order of MP>>DC>>>MO. LPS stimulation did not induce expression of SS or sst1,3,4,5. However, sst2 mRNA expression was upregulated significantly by stimulation with LPS. CST mRNA was upregulated as well. During differentiation of MO in MP or DC, time-dependent, significantly increasing sst2 and CST mRNA levels were found. By confocal microscopy, the presence of sst2 receptors was demonstrated on MP, but not on DC. This study demonstrates for the first time a selective and inducible expression of the recently discovered CST, as well as sst2, in human monocyte-derived cells, suggesting a role for a CST-sst2 system rather than a SS-sst2 system in these immune cell types.

Vgamma9Vdelta2 T cells recovered from eyes of patients with Behçet's disease recognize non-peptide prenyl pyrophosphate antigens. (Article)

2002-09-01T00:00:00Z

The phenotype and antigen-specificity of T cells expanded by mitogenic stimulation from intra-ocular fluid (IOF) samples of affected eyes of six Behçet's disease (BD) patients, and seven patients with other uveitis entities, were determined. High numbers of γδ T cells, predominantly Vγ9Vδ2 T cells, were only detected in the IOF-derived TCL of three BD patients. Whereas no TCL responded to heat shock protein (HSP) 65 kDa, reactivity to isopentyl pyrophosphate (IPP) and related non-peptide prenyl pyrophosphates (PPP) was restricted to the γδ T cell containing TCL. Upon IPP stimulation, these TCL secreted IFN-γ but no IL-4. By single-cell analysis of intracellular IFN-γ production and CD69 expression the IOF-derived IPP-specific T cells were identified as CD4−CD8− γδ T cells. The data presented suggest the infiltration of PPP-specific Vγ9Vδ2 Th1-like cells into the eye of BD patients with uveitis.

Somatostatin receptor subtypes in human thymoma and inhibition of cell proliferation by octreotide in vitro (Article)

2000-01-01T00:00:00Z

Somatostatin (SS) and SS receptor (SSR) subtypes, code-named sst1-5, are heterogeneously expressed in the normal human thymus. This suggests their involvement in controlling the immune and/or neuroendocrine functions in this organ. Moreover, recently a high in vivo uptake of [111In-DTPA-D-Phe1]octreotide has been reported in patients bearing thymoma. The present study characterizes in vivo and in vitro, functional SS-binding sites in a human thymoma. A high uptake of [111In-DTPA-D-Phe1]octreotide was observed in the chest of a patient with myasthenia gravis due to a cortical thymoma. Specific binding of [125I-Tyr11] SS-14 was found on a membrane preparation of the surgically removed thymoma. Scatchard analysis showed high affinity binding sites (Kd, 47.5 +/- 2.5 pmol/L) with low maximum binding capacity (23.5 +/- 2.5 fmol/mg membrane protein). RT-PCR analysis showed the presence of sst1, sst2A, and a predominant sst3 messenger RNA (mRNA) expression in the tumor tissue. Primary cultured tumor cells expressed sst3 mRNA only. In contrast to the normal thymus, SS mRNA was not expressed. By immunohistochemistry, the tumor cells highly expressed sst3 receptors, weakly expressed sst1 receptors, and showed no immunostaining for sst2A receptors. sst2A immunoreactivity was found in the stromal compartment of the tumor, particularly on the endothelium of small intratumoral blood vessels. In primary cultured tumor cells, both SS and octreotide (10 nmol/L) significantly inhibited [3H]thymidine incorporation by 40.6% and 43.2%, respectively. The following conclusions were reached. 1) As this tumor displayed a high immunoreactivity for sst3 and the cultured tumor cells expressed the sst3 mRNA only, this SSR may be the subtype involved in the inhibition of epithelial tumor cell proliferation by octreotide in vitro. 2) A loss of endogenous SS production in this thymoma might be implicated in the uncontrolled cell growth. 3) In this case, the sst3 may play a role in determining the uptake of [111In-DTPA-D-Phe1]octreotide by in vivo SS receptor scintigraphy.

Somatostatin receptor 2A expression in choroidal neovascularization secondary to age-related macular degeneration (Article)

2000-01-01T00:00:00Z

PURPOSE: The growth of ocular neovascularization is regulated by a balance between stimulating and inhibiting growth factors. Somatostatin affects angiogenesis by inhibiting the growth hormone-insulin-like growth factor axis and also has a direct antiproliferative effect on human retinal endothelial cells. The purpose of our study is to investigate the expression of somatostatin receptor (sst) subtypes and particularly sst subtype 2A (sst2A) in normal human macula, and to study sst2A in different stages of age-related maculopathy (ARM), because of the potential anti-angiogenic effect of somatostatin analogues. METHODS: Sixteen eyes (10 enucleated eyes, 4 donor eyes, and 2 surgically removed choroidal neovascular [CNV] membranes) of 15 patients with eyes at different stages of ARM were used for immunohistochemistry. Formaldehyde-fixed paraffin-embedded slides were incubated with a polyclonal anti-human sst2A antibody. mRNA expression of five ssts and somatostatin was determined in the posterior pole of three normal human eyes by reverse transcriptase-polymerase chain reaction. RESULTS: The immunohistochemical expression of sstA in newly formed endothelial cells and fibroblast-like cells was strong in fibrovascular CNV membranes. mRNA of sst subtypes 1, 2A, and 3, as well as somatostatin, was present in the normal posterior pole; sst subtypes 4 and 5 were not detectable. CONCLUSIONS: Most early-formed CNV in ARM express sst2A. The presence of mRNA of sst subtype 2A was observed in normal human macula, and subtypes 1 and 3 and somatostatin are also present. sst2A receptors bind potential anti-angiogenic somatostatin analogues such as octreotide. Therefore, somatostatin analogues may be an effective therapy in early stages of CNV in ARM.

Age-related decrease of somatostatin receptor number in the normal human thymus (Article)

2000-01-01T00:00:00Z

The thymus exhibits a pattern of aging oriented toward a physiological involution. The structural changes start with a steady decrease of thymocytes, whereas no major variations occur in the number of thymic epithelial cells (TEC). The data concerning the role of hormones and neuropeptides in thymic involution are equivocal. We recently demonstrated the presence of somatostatin (SS) and three different SS receptor (SSR) subtypes in the human thymus. TEC selectively expressed SSR subtype 1 (sst(1)) and sst(2A). In the present study we investigated whether SSR number is age related in the thymus. Binding of the sst(2)-preferring ligand (125)I-Tyr(3)-octreotide was evaluated in a large series of normal human thymuses of different age by SSR autoradiography and ligand binding on tissue homogenates. The score at autoradiography and the number of SSR at membrane homogenate binding (B(max)) were inversely correlated with the thymus age (r = -0.84, P < 0.001; r = -0.82, P < 0.001, respectively). The autoradiographic score was positively correlated with the B(max) values (r = 0.74, P < 0.001). Because the TEC number in the age range considered remains unchanged, the decrease of octreotide binding sites might be due to a reduction of sst(2A) receptor number on TEC. The age-related expression of a receptor involved mainly in controlling secretive processes is in line with the evidence that the major changes occurring in TEC with aging are related to their capabilities in producing thymic hormones. In conclusion, SS and SSR might play a role in the involution of the human thymus. These findings underline the links between the neuroendocrine and immune systems and support the concept that neuropeptides participate in development of cellular immunity in humans.

Somatostatin receptors in the haematopoietic system (Article)

2000-01-01T00:00:00Z

Somatostatin receptor subtypes in human immune cells (Article)

2000-01-01T00:00:00Z

Physiological and pathophysiological role of somatostatin receptors in the human thymus (Article)

2000-01-01T00:00:00Z

Somatostatin and somatostatin receptors in retinal diseases (Article)

2000-01-01T00:00:00Z

Refractory immune-mediated and haematological diseases: candidates for peptide receptor radiotherapy? (Article)

2000-01-01T00:00:00Z

In vitro characterization of somatostatin receptors in the human thymus and effects of somatostatin and octreotide on cultured thymic epithelial cells (Article)

1999-01-01T00:00:00Z

Somatostatin (SS) and its analogs exert inhibitory effects on secretive and proliferative processes of various cells via high affinity SS receptors (SS-R). SS analogs bind with different affinity to the five cloned SS-R subtypes. Octreotide, an octapeptide SS analog, binds with high affinity to the SS-R subtype 2 (sst2). SS-R have been demonstrated in vivo and in vitro on cells from endocrine and immune systems. Among the lymphatic tissues, the thymus has been shown to contain the highest amount of SS, suggesting a local functional role of the peptide. We investigated the SS distribution and SS-R expression pattern in the normal human thymus using autoradiography, membrane homogenate binding studies, and RT-PCR. In addition, the effect of SS and octreotide on growth of cultured thymic epithelial cells (TEC) was studied. By autoradiography, binding of [125I-Tyr0]-SS-28 and [125I-Tyr3]-octreotide was detected in all seven thymuses studied. Specific [125I-Tyr3]-octreotide binding was shown on membrane preparations from thymuses, while not from cultured thymocytes. RT-PCR showed the expression of sst1, sst2A and sst3 messenger RNA (mRNA) in the thymic tissue, whereas sst1 and sst2A mRNAs were found in isolated TEC. SS mRNA was present in thymic tissue and in isolated TEC. SS and octreotide significantly inhibited 3H-thymidine incorporation in 3 of 3 and 6 of 6 TEC cultures, respectively. The percent inhibition ranged from 38.8 to 66.8% for SS and from 19.1 to 59.5% for octreotide. In conclusion, SS mRNA and sst1, sst2A, and sst3 mRNAs are expressed in the normal human thymus. Cultured TEC selectively express sst1 and sst2A mRNA and respond in vitro to SS and octreotide administration with an inhibition of cell proliferation. These data suggest a paracrine/autocrine role of SS and its receptors in the regulation of cell growth in thymic microenvironment.

Somatostatin receptor subtype expression in cells of the rat immune system during adjuvant arthritis (Article)

1999-01-01T00:00:00Z

Somatostatin is a neuropeptide that is widely distributed throughout the body. It acts as a neurohormone and a neurotransmitter and may also have an immunomodulatory role. The genes for five subtypes of somatostatin receptors (sst) have been cloned, suggesting that the diverse effects of the peptide might be mediated by different receptors. We are interested in studying the role of sst ininflammation, using an animal model. Because of the up-regulation of sst expression in inflamed joints in human rheumatoid arthritis, we chose rat adjuvant arthritis as an experimental model. In order to determine which of the sst subtypes might be important in immune modulation, subtype expression in leukocytes isolated from different lymphoid tissues of the rat was studied. Also, the expression levels of the most abundantly expressed sst mRNAs in leukocytes from spleen and blood were compared in rats with adjuvantarthritis and controls, using a semi-quantitative approach. Furthermore, the effect of systemic administration of a long-acting somatostatin analogue, octreotide, which binds selectively to sst subtypes 2 and 5 (sst2 and sst5), on the incidence and the severity of rat adjuvant arthritis, was studied. The main sst expressed in cells of the rat immune system, both resting and activated, were found to be sst3 and sst4. This contrasts with the human and murine situations, in which sst2 appears to be the main subtype expressed in the immune system. No quantitative differences in sst subtype mRNA levels in leukocytes from spleen and blood were found between rats with adjuvant arthritis and controls. Finally, no effect of systemic administration of octreotide on either the incidence or severity of adjuvant arthritis in Lewis rats was found. As octreotide binds selectively to sst2 and sst5, the absence of an immunomodulatory effect of this analogue in rat adjuvant arthritis corroborates our finding that these sst subtypes are not expressed in cells of the rat immune system. In conclusion, cells of the rat immune system appear to express a spectrum of sst (sst3 and sst4) different from that found in human granulomatous and autoimmune disease (mainly sst2). Therefore, the rat adjuvant arthritis model appears to be suitable only for studying the immunomodulatory effects of somatostatin analogues which have a high affinity for sst3 and sst4, but not for studying the immunomodulatory effects of octreotide, which has a high affinity only for sst2 and sst5.

Somatostatin-receptor scintigraphy in Graves' orbitopathy (Article)

1996-07-02T00:00:00Z

Somatostatin and substance P analogues: applications in autoimmune and haematological diseases (Doctoral Thesis)

1995-12-13T00:00:00Z

Somatostatin has been extensively studied in relation to the endocrine and nervous systems. Many reports on the role of somatostatin receptor imaging and somatostatin treatment of neuroendocrine tumours have been published. The relation between somatostatin and other neuropeptides and the immune system is less explored. The aim of this study was to investigate the diagnostic applications of somatostatin and substance P analogues in autoimmune and haematological diseases. Both in vivo and in vitro studies were performed, using peptide receptor scintigraphy in patients and rats, peptide receptor autoradiography on tissue biopsies, ligand binding assays on cell homogenates, and polymerase chain reactions on lymphoid cell lines. Moreover, based on the results of these studies, speculations were made about the therapeutical applications of somatostatin and substance P analogues in autoimmune and haematological diseases.