Notch pathway inhibition controls myeloma bone disease in the murine MOPC315.BM model.

Despite evidence that deregulated Notch signalling is a master regulator of multiple myeloma (MM) pathogenesis, its contribution to myeloma bone disease remains to be resolved. Notch promotes survival of human MM cells and triggers human osteoclast activity in vitro. Here, we show that inhibition of Notch through the γ-secretase inhibitor XII (GSI XII) induces apoptosis of murine MOPC315.BM myeloma cells with high Notch activity. GSI XII impairs murine osteoclast differentiation of receptor activator of NF-κB ligand (RANKL)-stimulated RAW264.7 cells in vitro. In the murine MOPC315.BM myeloma model GSI XII has potent anti-MM activity and reduces osteolytic lesions as evidenced by diminished myeloma-specific monoclonal immunoglobulin (Ig)-A serum levels and quantitative assessment of bone structure changes via high-resolution microcomputed tomography scans. Thus, we suggest that Notch inhibition through GSI XII controls myeloma bone disease mainly by targeting Notch in MM cells and possibly in osteoclasts in their microenvironment. We conclude that Notch inhibition is a valid therapeutic strategy in MM.

Education research: cognitive performance is preserved in sleep-deprived neurology residents.

OBJECTIVE: To test the hypotheses that sleep deprivation in neurology residents is associated with performance deficits and that vigilance and cognitive performance is more compromised after overnight on-call duty compared to night shift. METHODS: Thirty-eight neurology residents of a university teaching hospital participated in a prospective single-blind comparison study. Residents were recruited according to their working schedule and divided into 3 groups: 24 hours overnight on-call duty, night shift, and regular day shift (controls). All participants underwent serial measurements of sleepiness and cognitive performance in the morning directly after or before their shift. Pupillary sleepiness test and Paced Auditory Serial Addition Test were applied. Perceived sleepiness was assessed by a questionnaire. RESULTS: Sleepiness was increased in residents after night shift and overnight call compared to controls while the type of night duty was not associated with the extent of sleepiness. Sleep-deprived residents did not show any performance deficits on the Paced Auditory Serial Addition Test. Cognitive performance was not associated with sleepiness measures. CONCLUSIONS: Night shift and overnight call duty have a similar impact on alertness in neurology residents. Sleep-deprived neurology residents may be able to overcome sleep loss-related performance difficulties for short periods.

CXCR3+CD4+ T cells are enriched in inflamed kidneys and urine and provide a new biomarker for acute nephritis flares in systemic lupus erythematosus patients.

OBJECTIVE: The high frequency of CD4+ T cells in interstitial infiltrates of patients with lupus nephritis suggests a contribution of these cells to local pathogenesis. The aim of this study was to examine the role of CXCR3 and the chemokine CXCL10 in recruiting these cells into the kidney and to determine whether the infiltrating T cells could be monitored in the urine to provide a reliable biomarker for acute lupus nephritis. METHODS: The frequencies of CD3+ T cells, CXCR3+ cells, and CXCL10+ cells were determined by immunohistochemical and immunofluorescence analyses of kidney sections from 18 patients with lupus nephritis. The frequency of CXCR3+CD4+ T cells was determined by flow cytometry of peripheral blood and urine from 38 patients with systemic lupus erythematosus (SLE), and the values were compared with disease activity as determined by the Systemic Lupus Erythematosus Disease Activity Index. RESULTS: In renal biopsy tissues from patients with lupus nephritis, a mean of 63% of the infiltrating cells expressed CXCR3, approximately 60% of them were T cells, and the CXCR3+ cells colocalized with CXCL10-producing cells. In biopsy tissues from SLE patients with acute nephritis, approximately 50% of the urinary CD4+ T cells were CXCR3+, as compared with 22% in the peripheral blood, and the frequency of urinary CXCR3+CD4+ T cells correlated with disease activity. Moreover, the number of urinary CD4+ T cells reflected nephritis activity, and elevation above 800 CD4+ T cells per 100 ml of urine sharply delineated active from inactive nephritis. CONCLUSION: CXCR3+ T cells are recruited into the inflamed kidneys, are enriched in the urine, and are a valuable marker of nephritis activity in SLE. They also present a potential target for future therapies.

Human mastoid periosteum-derived stem cells: promising candidates for skeletal tissue engineering.

Currently, mesenchymal stem cells (MSCs) are considered as the most eligible cells for skeletal tissue engineering. However, factors such as difficult stimulation and control of differentiation in vivo hamper their clinical use. In contrast, periosteum or periosteum-derived cells (PCs) are routinely clinically applied for bone and cartilage repair. PCs have often been named MSCs but, although cells of osteochondrogenic lineages arise from MSCs, it is unclear whether periosteum really contains MSCs. Our aim was to investigate the MSC-like character of PCs derived from the periosteum of mastoid bone. Harvesting of periosteum from mastoid bone is easy, so mastoid represents a good source for the isolation of PCs. Therefore, we analysed the MSC-like growth behaviour and the expression of embryonic, ectodermal, endodermal and mesodermal markers by microarray and FACS technology, and the multilineage developmental capacity of human PCs. Regarding clinical relevance, experiments were performed in human serum-supplemented medium. We show that PCs do not express early embryonic stem cell markers such as Oct4 and Nanog, or the marker of haematopoietic stem cells CD34, but express some other MSC markers. Osteogenesis resulted in the formation of calcified matrix, increased alkaline phosphatase activity, and induction of the osteogenic marker gene osteocalcin. Staining of proteoglycans and deposition of type II collagen documented chondrogenic development. As shown for the first time, adipogenic stimulation of mastoid-derived PCs resulted in the formation of lipid droplets and expression of the adipogenic marker genes aP2 and APM1. These results suggest MSC-like PCs from mastoid as candidates for therapy of complex skeletal defects.

Bortezomib inhibits human osteoclastogenesis.

In multiple myeloma, the overexpression of receptor activator of nuclear factor kappa B (NF-kappaB) ligand (RANKL) leads to the induction of NF-kappaB and activator protein-1 (AP-1)-related osteoclast activation and enhanced bone resorption. The purpose of this study was to examine the molecular and functional effects of proteasome inhibition in RANKL-induced osteoclastogenesis. Furthermore, we aimed to compare the outcome of proteasome versus selective NF-kappaB inhibition using bortezomib (PS-341) and I-kappaB kinase inhibitor PS-1145. Primary human osteoclasts were derived from CD14+ precursors in presence of RANKL and macrophage colony-stimulating factor (M-CSF). Both bortezomib and PS-1145 inhibited osteoclast differentiation in a dose- and time-dependent manner and furthermore, the bone resorption activity of osteoclasts. The mechanisms of action involved in early osteoclast differentiation were found to be related to the inhibition of p38 mitogen-activated protein kinase pathways, whereas the later phase of differentiation and activation occurred due to inhibition of p38, AP-1 and NF-kappaB activation. The AP-1 blockade contributed to significant reduction of osteoclastic vascular endothelial growth factor production. In conclusion, our data demonstrate that proteasomal inhibition should be considered as a novel therapeutic option of cancer-induced lytic bone disease.

Immunological memory stabilizing autoreactivity.

The etiopathologies of autoimmune diseases are complex. A broad variety of cell types and gene products are involved. However, clinical and experimental evidence suggests that the importance of an individual factor changes during the course of the disease. Factors and cell types that induce acute autoreactivity and initiate an autoimmune disease could be distinct from those that drive a chronic course of that disease. Autoreactive immunological memory, in particular B cell and plasma cell memory, contributes to chronicity through several mechanisms. Formation of autoreactive memory B cells leads to an increase in the numbers of autoreactive cells. In comparison to naive B cells, these memory B cells show a decreased threshold for activation. Additionally, a fraction of memory B cells express the chemokine receptor CXCR3, which supports their accumulation within chronically inflamed tissues. This may allow their escape from mechanisms for induction of peripheral tolerance. Within the inflamed tissue, inflammatory cytokines and autoantigens provide activation signals that promote plasma cell differentiation and survival. The autoantibodies produced locally by these plasma cells contribute to the severity of inflammation. Together, an autoreactive loop of autoantibody-induced inflammation is formed. Another integral part of immunological memory are long-lived plasma cells. These cells provide persistent humoral antibody memory. Though not all autoantibodies are produced by long-lived plasma cells, these cells have a special impact on immune pathology. Long-lived plasma cells are relatively resistant to existing therapies of immunosuppression and continuously secrete antibodies, without need for restimulation. Long-lived plasma cells provide titers of autoantibodies even during clinically quiescent phases and after immunosuppression. These persisting autoantibody titers, though often low and not causing acute clinical symptoms, are likely to maintain a low level of chronic inflammation and progressive tissue destruction, which reduces the threshold for another break of immunological tolerance.

Induction of pathogenic anti-dsDNA antibodies is controlled on the level of B cells in a non-lupus prone mouse strain.

The SmD1(83-119) peptide is a main target of autoantibodies and T cells in human and murine lupus, but its role in autoimmunity induction remains elusive. Therefore, female Balb/c mice and (NZW x Balb/c)F1 [CWF1] mice with identical MHC haplotype as lupus prone NZB/W mice were immunized with SmD1(83-119). Immunizations of CWF1 mice with SmD1(83-119), but not with the controls (irrelevant peptide, HEL peptide, or saline), induced anti-SmD1(83-119) and anti-dsDNA antibodies and proteinuria not present in Balb/c mice. DsDNA-specific plasma cell induction after SmD1(83-119) immunizations was confirmed by ELISPOT assays showing that the generation of dsDNA-specific antibody forming cells (AFC) was mainly driven by increased T-cell help. T-cell help for the generation of dsDNA-specific AFC was also present in saline-treated CWF1 mice but was controlled on the levels of B cells preventing autoimmunity.

Detection and characterization of plasma cells in peripheral blood: correlation of IgE+ plasma cell frequency with IgE serum titre.

In atopic patients and patients with hyper-IgE syndrome (HIE) highly elevated IgE serum levels can be detected. Due to their very low frequency little is known about IgE-producing plasma cells (PC) in peripheral blood. We used CD138 MACS microbeads to enrich plasma cells from peripheral blood of normal donors, atopic patients and one HIE patient. CD138+ cells were mainly CD45+, CD44++, CD19dim, CD38++, CD27++, CD86+, HLA-DR+/++, CD71dim, VLA-4+, VLA-5-, CD28-, CD25-, CD69-, CLA-, CD20-, CD21- and CD22-. They show weak expression of surface Ig but high levels of intracellular Ig and they secrete Ig in culture. Thus CD138+ cells from peripheral blood show characteristics of early plasma cells. IgE+ CD138+ plasma cells could be detected in 19 of 24 normal donors with an average frequency of 0.06% IgE+ cells among CD138+ cells. Higher frequencies were detected in atopic patients, atopic patients with markedly elevated serum IgE levels and the hyper-IgE patient with an average of 0.32%, 7.21% and 6.54%, respectively. Additionally, using the recently developed cellular affinity matrix technology, we were able to detect IgE secreting plasma cells and thereby could demonstrate that most of the IgE secreting cells express CD138. The frequency of IgE+ CD138+ cells among PBMC correlated highly significantly with serum IgE titres (r = 0.8532***), indicating that IgE secreting CD138+ cells in peripheral blood are directly related to the plasma cell pool contributing to the IgE titre.

Strong acceleration of murine lupus by injection of the SmD1(83-119) peptide.

OBJECTIVE: The mechanisms of IgG anti-double-stranded DNA (anti-dsDNA) antibody induction are incompletely understood. We recently demonstrated a high prevalence of autoantibodies to the C-terminus of SmD1 in patients with systemic lupus erythematosus (SLE) that was closely associated with anti-dsDNA reactivity. The aim of the present study was to analyze the influence of the SmD1 C-terminus on the generation of pathogenic anti-dsDNA antibodies in a murine model of SLE. METHODS: Female lupus-prone prenephritic (NZB x NZW)F1 mice (NZB/NZW mice) as well as female control BALB/c, NZW, and (BALB/c x NZW)F, mice (CWF1 mice) were subcutaneously injected with keyhole limpet hemocyanin (KLH)-coupled SmD1(83-119). Controls received injections of recombinant SmD1 (rSmD1), KLH-rSmD1, KLH-coupled randomized peptide of SmD1(83-119), ovalbumin, or saline. Animals were monitored for survival and proteinuria and for levels of plasma creatinine, urea, and autoantibodies. In addition, histologic examinations were performed and T cell responses against SmD1(83-119) peptide and rSmD1 protein were determined in SmD1(83-119)-treated and -untreated NZB/NZW mice. RESULTS: Immunization with KLH-SmD1(83-119), but not with control peptide, significantly accelerated the natural course of lupus in NZB/NZW mice, with premature renal failure and increased development of anti-dsDNA antibodies. Control strains of mice remained healthy, with no relevant anti-SmD1(83-119) antibodies detectable even after immunization. In contrast to findings in control mice, a T cell response against SmD1(83-119) was already present in unmanipulated NZB/NZW mice, and this response was further amplified after immunization. CONCLUSION: The SmD1(83-119) peptide can influence the pathogenic anti-dsDNA response in the NZB/NZW murine lupus model. The data suggest that an SmD1(83-119)-specific T cell response is critical. Therefore, modulation of these autoantigen-specific T cells by tolerance induction may provide a therapeutic approach to specific immunosuppression in lupus.

Inflamed kidneys of NZB / W mice are a major site for the homeostasis of plasma cells.

(NZB x NZW)F1 (NZB / W) mice develop a disease similar to human systemic lupus erythematosus (SLE), including autoantibody production, hypergammaglobulinaemia and inflammation of the kidneys. It is known that large numbers of lymphocytes infiltrate the kidneys of these mice. Here, we compare the roles of bone marrow, spleen and inflamed kidneys of NZB / W mice in the activation of B cells and the persistence of antibody-secreting cells (ASC). ASC are present in the kidneys of NZB / W mice with full-blown disease, as many as in the spleen and bone marrow. The specificity of the ASC in the inflamed kidneys is not restricted to self-antigens. After immunization of NZB / W mice with ovalbumin (OVA) the OVA-specific ASC are found initially in the spleen. Weeks later, OVA-specific ASC are found in high numbers in the bone marrow and the kidneys of these mice, but no longer in the spleen. As determined by FACS, B cells with a germinal center phenotype (B220(+) / PNA(+)) are found only in very low numbers in the kidneys, but in high numbers in the spleen of NZB / W mice. Germinal centers could not be detected in the kidneys, but in the spleen, and plasma cells appear to be scattered over the tissue. These data suggest that in autoimmune NZB / W mice, plasma cells generated in immune reactions of secondary lymphoid organs, later accumulate and persist in the inflamed kidneys, were they enhance the local concentrations of Ab and immunocomplexes. These experiments identify the inflamed kidneys of NZB / W mice as a site of prime relevance for the homeostasis of plasma cells, irrespective of their specificity.

Anxious and depressive symptoms in adolescents: epidemiological data of a large scale study in Dresden.

OBJECTIVES: The present large scale epidemiological study was designed to assess the prevalence of mental disorders in adolescents. METHODS: Two cross-sectional studies have been performed in adolescents in Dresden and the results of the examination of 627 high school and 485 secondary school students (mean age 15.5 years) are presented. Self rating procedures like the Beck Depression inventory (BDI) and clinical interviews have been used to enhance validity. RESULTS: The results underline the high prevalence rates of anxiety and depression in adolescents. Up to 30% of the 9th and 10th grades students suffer from mental disorders or are at risk for the development of mental disorders. CONCLUSIONS: Therefore primary prevention of mental disorders is desirable.

[Anxiety and depression in students. A study in Dresden middle schools and high schools]. / Angst und Depression bei Schülern. Eine Untersuchung an Dresdner Mittelschulen und Gymnasien.

OBJECTIVES: Recent investigations by the WHO have shown once again the high prevalence of mental disorders in European countries. Mainly in the field of mental disorders considerable deficits exist particularly with regard to primary prevention. METHODS: Although there is no doubt as to the importance of primary prevention within the scope of Public Health, there are deficits in this branch in Germany. This is unfortunate, considering that the proof of successful interventions is evidence for causal risk models. The current investigation is part of a study focusing upon the development, implementation and evaluation of a prevention program for anxiety and depression in adolescents and young adults. RESULTS: Presented here are the prevalence rates of anxiety disorders and depression ascertained in the first cross-sectional study carried out in secondary schools and high schools. A total of 627 high school students and 485 secondary school students in grades nine and ten in Dresden were examined.

Enrichment and detection of live antigen-specific CD4(+) and CD8(+) T cells based on cytokine secretion.

Following appropriate antigen-specific stimulation, CD4(+) and CD8(+) T lymphocytes rapidly express cytokines. Based on this stimulation-induced cytokine secretion and using cell surface affinity matrix technology we have developed a new method that permits specific, rapid and efficient detection, isolation and characterization of live antigen-specific CD4(+) and CD8(+) T lymphocytes. The power of this technique is demonstrated here for HLA-A0201-restricted influenza matrix protein peptide 58-66-specific CD8(+) cytotoxic T lymphocytes, influenza A virus- and recombinant tetanus toxin C fragment-specific Th1 cells and tetanus toxoid-specific Th2 cells.

Cerebral endothelial cells release TNF-alpha after stimulation with cell walls of Streptococcus pneumoniae and regulate inducible nitric oxide synthase and ICAM-1 expression via autocrine loops.

TNF-alpha, inducible NO synthase (iNOS), and ICAM-1 are considered to be key proteins in the inflammatory response of most tissues. We tested the hypothesis that cell walls of Streptococcus pneumoniae (PCW), the most common cause of adult bacterial meningitis, induce TNF-alpha, iNOS, and ICAM-1 expression in rat primary brain microvascular endothelial cell cultures. We detected TNF-alpha mRNA by RT-PCR already 1 h after stimulation with PCW, while TNF-alpha protein peaked at 4 h (9.4 +/- 3.6 vs 0.1 +/- 0.1 pg/microgram protein). PCW induced iNOS mRNA 2 h after stimulation, followed by an increase of the NO degradation product nitrite (18.1 +/- 4 vs 5.8 +/- 1.8 at 12 h; 18.1 +/- 4 vs 5.8 +/- 1.8 pmol/microgram protein at 72 h). The addition of TNF-alpha Ab significantly reduced nitrite production to 62.2 +/- 14.4% compared with PCW-stimulated brain microvascular endothelial cells (100%). PCW induced the expression of ICAM-1 (measured by FACS), which was completely blocked by TNF-alpha Ab (142 +/- 18.6 vs 97.5 +/- 12.4%; 100% unstimulated brain microvascular endothelial cells). Cerebral endothelial cells express TNF-alpha mRNA as well as iNOS mRNA and release the bioactive proteins in response to PCW. PCW-induced NO production is mediated in part by an autocrine pathway involving TNF-alpha, whereas ICAM-1 expression is completely mediated by this autocrine loop. By these mechanisms, cerebral endothelial cells may regulate critical steps in inflammatory blood-brain-barrier disruption of bacterial meningitis.

Survival of long-lived plasma cells is independent of antigen.

Recent studies have shown that persistent specific antibody titer is provided by long-lived plasma cells (PC) which constitute a new kind of 'memory-providing cells'. In the present study, we examine the role of antigen for the long-term survival of PC and the maintenance of specific serum antibody titers. Using a novel cytometric technology, to identify and isolate antigen-specific PC, we analyzed long-lived PC of BALB/c mice, during their development (between day 1 and 10) after secondary immunization with ovalbumin (OVA) and in the phase of the established immune reaction. Most if not all OVA-specific PC were generated within a few days after immunization. Within approximately 3 weeks, they matured, as indicated by down-regulation of expression of MHC class II. These PC are long lived and located in spleen and bone marrow. Upon adoptive transfer, OVA-specific PC from bone marrow, but not memory B cells, conferred specific and long-lasting antibody titers to antigen-free IgH syngeneic recipients. In response to antigenic challenge, new OVA-specific antibody-secreting cells were generated from transferred memory B cells. Antibody secretion by long-lived PC was not affected. Our results confirm that persistent antibody titers are provided by long-lived PC, independent of memory B cells and demonstrate that this humoral memory is inert to antigen.

Analysis and sorting of T cells according to cytokine expression.

Functional distinct populations of T helper cells can be defined according to the expression of cytokines. A remarkable diversity of cytokine expression has been demonstrated in single T cells even from clonal populations and up to now no stable surface markers have been described which on the single-cell level directly correlate with the secretion of a certain cytokine. Since cytokines are the major parameters of T cell effector function we have developed strategies which now allow to separate cells according to the specific cytokines they secrete. The "affinity matrix technology"--secreted molecules are relocated to the cell surface by an artificially created antibody matrix--allows to isolate cells according to a distinct secreted product. In addition to this universal, but laborious technology, we could demonstrate by high-sensitivity immunofluorescence that IFN-gamma and IL-10 but not IL-2 and IL-4 are specifically expressed in low copy number on the surface of cells secreting these cytokines. Surface IFN-gamma and IL-10 are the first unambiguous surface markers for pro-inflammatory IFN-gamma-secreting Th 0/1 cells and IL-10-producing anti-inflammatory Th2/3 cells. We have used purified cytokine-secreting T cells to study in individual T cells the sequential production of IL-2, IFN-gamma, and IL-10, the stability of IFN-gamma expression and the selective homing of IFN-gamma-producing cells into inflamed tissues.