Philipp von Landenberg

Vitamin D and musculoskeletal health, cardiovascular disease, autoimmunity and cancer: Recommendations for clinical practice

BACKGROUND There is increasing evidence that, in addition to the well-known effects on musculoskeletal health, vitamin D status may be related to a number of non-skeletal diseases. An international expert panel formulated recommendations on vitamin D for clinical practice, taking into consideration the best evidence available based on published literature today. In addition, where data were limited to smaller clinical trials or epidemiologic studies, the panel made expert-opinion based recommendations. METHODS Twenty-five experts from various disciplines (classical clinical applications, cardiology, autoimmunity, and cancer) established draft recommendations during a 2-day meeting. Thereafter, representatives of all disciplines refined the recommendations and related texts, subsequently reviewed by all panelists. For all recommendations, panelists expressed the extent of agreement using a 5-point scale. RESULTS AND CONCLUSION Recommendations were restricted to clinical practice and concern adult patients with or at risk for fractures, falls, cardiovascular or autoimmune diseases, and cancer. The panel reached substantial agreement about the need for vitamin D supplementation in specific groups of patients in these clinical areas and the need for assessing their 25-hydroxyvitamin D (25(OH)D) serum levels for optimal clinical care. A target range of at least 30 to 40 ng/mL was recommended. As response to treatment varies by environmental factors and starting levels of 25(OH)D, testing may be warranted after at least 3 months of supplementation. An assay measuring both 25(OH)D(2) and 25(OH)D(3) is recommended. Dark-skinned or veiled individuals not exposed much to the sun, elderly and institutionalized individuals may be supplemented (800 IU/day) without baseline testing.

Mechanism of lipid-body formation in prokaryotes: how bacteria fatten up.

Neutral lipid accumulation is frequently observed in some Gram‐negative prokaryotes like Acinetobacter sp. and most actinomycetes, including the pathogenic Mycobacterium tuberculosis and antibiotic producing streptomycetes. We examined the formation  of  wax  ester‐  and  triacylglycerol  (TAG)‐bodies in Acinetobacter calcoaceticus and Rhodococcus opacus using microscopic, immunological and biophysical methods. A general model for prokaryotic lipid‐body formation is proposed, clearly differing from the current models for the formation of lipid inclusions in eukaryotes and of poly(hydroxyalkanoic acid) (PHA) inclusions in prokaryotes. Formation of lipid‐bodies starts with the docking of wax ester synthase/acyl‐CoA:diacylglycerol acyltransferase (WS/DGAT) to the cytoplasm membrane. Both, analyses of in vivo and in vitro lipid‐body synthesis, demonstrated the formation of small lipid droplets (SLDs), which remain bound to the membrane‐associated enzyme. SLDs conglomerated subsequently to membrane‐bound lipid‐prebodies which are then released into the cytoplasm. The formation of matured lipid‐bodies in the cytoplasm occurred by means of coalescence of SLDs inside the lipid prebodies, which are surrounded by a half‐unit membrane of phospholipids.

The VP1 Unique Region of Parvovirus B19 and Its Constituent Phospholipase A2-Like Activity

ABSTRACT Parvovirus B19 is the causative agent of erythema infectiosum. In addition, parvovirus B19 infection may be associated with other disease manifestations, namely, thrombocytopenia or granulocytopenia, spontaneous abortion or hydrops fetalis in pregnant women, acute and chronic arthritis, and systemic lupus erythematosus. Based on sequence homology data, a phospholipase A2 motif has been identified in the VP1 unique region of parvovirus B19. (Y. Li et al., J. Gen. Virol. 82:2821-2825, 2001; Z. Zadori et al., Dev. Cell 1:291-302, 2001). We have established a new in vitro assay based on electrospray ionization tandem mass spectroscopy to show that phospholipase A2 activity is present in the VP1 unique region produced in Escherichia coli and in virus-like particles consisting of combinations of VP1 and VP2 proteins expressed by recombinant baculovirus. The enzyme activity of the VP1 unique region showed typical Ca2+ dependency and could be inhibited by manoalide and 4-bromophenacylbromide, which bind covalently to lysine and histidine residues, respectively, as part of the active center of the enzyme. By using subfragments, we demonstrated an association between the phospholipase A2-like activity and the carboxy-terminal domain of the VP1 unique region.

Parvovirus B19 infection and autoimmune disease.

Human parvovirus B19 infections may cause a widespread benign and self-limiting disease in children and adults, known as erythema infectiosum or fifth disease. A variety of further manifestations are associated with the infection such as arthralgias, arthritis, leukopenia and thrombocytopenia, anemia and vasculitis, spontaneous abortion and hydrops fetalis in pregnant women. Both in children and adults parvovirus B19 infections have been frequently implicated as a cause or trigger of various forms of autoimmune diseases affecting joints, connective tissue and large and small vessels. In addition, autoimmune neutropenia, thrombocytopenia and hemolytic anemia are known as sequelae of B19 infection. The molecular basis of the autoimmune phenomena and resultant pathogenesis is unclear. The involvement of molecular mimicry between cellular and viral proteins, the induction of enhanced cytokine production via the viral transactivator protein NS1 and the phospholipase A2-like activity of the capsid protein VP1 may contribute to the induction of autoimmune reactions. All the known data and the potential mechanisms involved in the pathogenesis will be discussed in this review.

Interaction of TLR2 and TLR4 Ligands with the N-terminal Domain of Gp96 Amplifies Innate and Adaptive Immune Responses

Activation of dendritic cells by ligands for Toll-like receptors (TLR) is a crucial event in the initiation of innate and adaptive immune responses. Several classes of TLR ligands have been identified that interact with distinct members of the TLR-family. TLR4 ligands include lipopolysaccharide derived from different Gram-negative bacteria and viral proteins. Recent reports have demonstrated the TLR-mediated activation of dendritic cells by heat shock proteins (HSPs). However, doubts were raised as to what extent this effect was due to lipopolysaccharide contaminations of the HSP preparations. We re-examined this phenomenon using Gp96 or its N-terminal domain, nominally endotoxin-free (<0.5 enzyme units/mg). As described previously, innate immune cells are activated by Gp96 at high concentrations (≥50 μg/ml) but not at lower concentrations. However, preincubation of low amounts of Gp96 with TLR2 and TLR4 ligands at concentrations unable to activate dendritic cells by themselves results in the production of high levels of proinflammatory cytokines, up-regulation of activation markers, and amplification of T cell activation. Our results provide significant new insights into the mechanism of HSP-mediated dendritic cell activation and present a new function of HSPs in the amplification of dendritic cell activation by bacterial products and induction of adaptive immune responses.

Toll-like receptors and autoimmunity

The understanding of autoimmune diseases experienced an impressive boost since the Toll-like receptors (TLRs) have been identified as possible key players in autoimmune pathophysiology. Although these receptors recognize a variety of structures derived from viruses, bacteria and fungi leading to subsequent initiation of the relevant immune responses recent data support the idea that TLRs are crucial in the induction and perpetuation of certain autoimmune diseases, especially the systemic lupus erythematosus (SLE). In this review we will summarize recent data on involvement of TLRs in the development of autoimmune diseases. This review will focus on TLRs 7, 8 and 9 which were originally identified as receptors specific for bacterial and viral RNA/DNA, but more recent in vitro and in vivo studies have linked these receptors to the detection of host RNA, DNA, and RNA- or DNA-associated proteins in the context of autoimmunity.

Autoantibody Detection Using Indirect Immunofluorescence on HEp-2 Cells

The detection of autoantibodies is an important element in the diagnosis and monitoring of disease progression in patients with autoimmune diseases. In laboratory diagnostic tests for connective tissue and autoimmune liver diseases, indirect immunofluorescence on HEp‐2 cells plays a central role in a multistage diagnostic process. Despite the high quality of diagnostics, findings at different laboratories can differ considerably due to a lack of standardization, as well as subjective factors.

Antiphospholipid antibodies induce translocation of TLR7 and TLR8 to the endosome in human monocytes and plasmacytoid dendritic cells.

The antiphospholipid syndrome (APS) is an autoimmune disease characterized by thromboembolic events and/or fetal loss in the presence of antiphospholipid antibodies (aPLs). The mechanisms underlying the pathogenicity of aPLs are still poorly understood. Here we show that 3 human monoclonal aPLs as well as IgG fractions from patients with the APS increase mRNA expression of the intracellular toll-like receptor (TLR) 7 in plasmacytoid dendritic cells and TLR8 in monocytes. Simultaneously they induce the translocation of TLR7 or TLR8 from the endoplasmic reticulum to the endosome. These effects depend on the uptake of aPLs into the endosome, subsequent activation of endosomal NADPH oxidase, and generation of superoxide. As a consequence cells are dramatically sensitized to ligands for TLR7 and TLR8. This observation delineates a novel signal transduction pathway in innate immunity originating from the endosome. Because the overexpression of TLR7 can also be detected in plasmacytoid dendritic cells from patients with the APS ex vivo, our results provide an explanation for proinflammatory and procoagulant effects of aPLs. Because inappropriate expression of TLR7 has been implicated in the development of systemic autoimmunity, these findings may also be relevant for the understanding of autoimmunity.

Human antiphospholipid antibodies induce TNFα in monocytes via Toll-like receptor 8

The antiphospholipid syndrome (APS) is characterized by recurrent arterial and/or venous thromboses, pregnancy loss and the presence of antiphospholipid antibodies (aPL). One of the discussed mechanisms of this thrombotic activity in APS patients is attributed to TNFalpha secretion in monocytes after aPL stimulation. To investigate this mechanism in detail, we employed a monoclonal aPL and IgG fractions of APS patients for stimulation of human peripheral monocytes. Stimulation with this monoclonal aPL resulted in an increased expression and secretion of TNFalpha, caused by specific upregulation of TLR8 mRNA and protein expression levels. To confirm the specificity of this finding we could demonstrate that the TNFalpha enhancement could be neutralized by a TLR8-specific inhibitory DNA-oligonucleotide and could be further increased by adding the specific ligands for TLR8. Using APS patients IgG fractions for stimulation of peripheral monocytes revealed a similar TLR8 mRNA elevation and increase in TNFalpha-production. Furthermore the TLR8 expression level in PBMCs of APS patients was as well significantly elevated. It could be demonstrated that the TNFalpha release in monocytes resulting from aPL stimulation was exclusively induced by TLR8 engagement. This could be confirmed in PBMCs of APS patients, hinting that endogenous stimulation of TLR8 in APS patients and consecutive elevation of TNFalpha promotes a proinflammatory environment.

Interaction of inflammation, thrombosis, aspirin and enoxaparin in CNS experimental antiphospholipid syndrome.

Experimental antiphospholipid syndrome (eAPS) induced by immunization with beta(2)-glycoprotein I (beta(2)-GPI) causes behavioral hyperactivity. We assessed the role of thrombotic and inflammatory perivascular factors and standard APS therapies for CNS manifestations. Groups of mice (n=10 per group) were immunized once with beta(2)-GPI (eAPS) or adjuvant (controls) and treated daily from 1 month after immunization with either sham injections, aspirin (1.2 mg/kg) or enoxaparin (1 mg/kg) for 3 months. Serum antiphospholipid antibodies (aPL) and brain levels of tissue necrosis factor-alpha (TNF-alpha) and prostaglandin E (PGE) were then measured by ELISA and thrombin inhibitors by immunoblot. Behavioral hyperactivity was assessed by the staircase test. The eAPS mice had higher levels of aPL than adjuvant immunized controls. Inflammatory markers were found to be twofold higher and intrinsic brain thrombin inhibitors 50% lower in eAPS brains compared to controls. aPL titers were unaffected by treatment. Both aspirin and enoxaparin normalized brain concentrations of PGE and TNF-alpha and elevated thrombin inhibitors, the latter effect being more pronounced for enoxaparin. The increased activity and rearing exploratory behavior in eAPS (138.6+/-13.6 and 141.9+/-13.9% of controls, respectively) were attenuated significantly more by treatment with enoxaparin (91.5+/-12.3 and 95.0+/-9.8%) than by aspirin (167.0+/-18.4 and 114.7+/-13.1%, p<0.01, ANOVA). Together, these results demonstrate that eAPS is associated with significant brain inflammation and thrombosis that are well treated with both standard therapies for human APS. Enoxaparin attenuates behavior better than aspirin possibly by reducing thrombosis or stabilization of the blood brain barrier, suggesting an advantage for similar drugs in CNS APS.