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The proinflammatory protease caspase-1 plays pivotal roles in central pathways of innate immunity, thereby contributing to pathogen clearance. Beside its physiological role, dysregulated activity of caspase-1 is known to contribute to an increasing number of diseases. In this study, we optimized and validated a low-volume human whole blood assay facilitating the measurement of caspase-1 activation and inflammasome-related gene expression upon stimulation of the NLRP3, NLRC4 or AIM2 inflammasome. Using the NLRP3 inflammasome specific inhibitor MCC950, we were able to measure the activity of canonical or alternative NLRP3 pathways, AIM2 and NLRC4 inflammasomes in whole blood. Based on our data we assume a superposition of NLRP3 and NLRC4 inflammasome activities in human whole blood following stimulation with S. typhimurium. The optimized whole blood assay may be suitable for diagnostic and research purposes for pediatric patients who can only donate small amounts of blood.

A growing number of studies show an association between seasonal allergic rhinitis (SAR) with depression and anxiety. The underlying mechanisms of a link between SAR and affect, however, are still unclear. The objective of the present study was to investigate depressive symptoms and anxiety in SAR patients and their association to inflammatory and endocrine parameters. SAR patients (n=41) and non-allergic, healthy controls (n=42) were assessed during (pollen season) and out of symptomatic periods (non-pollen season). Inflammatory cytokine profile (Interleukin [IL]-2, IL-4, IL-6, IL-8, IL-10, IL-17, IFN-γ, TNF-α), Immunoglobulin-E (IgE), hair cortisol concentrations (HCC), as well as sleep quality were measured. The present data show that during acute allergic inflammation SAR patients experienced a significant increase in Beck Depression Inventory (BDI-) II scores when (a) compared to the asymptomatic period and (b) when compared to the non-allergic controls, while no differences in anxiety were observed. Increased BDI-II scores in SAR patients were significantly associated with levels of IL-6 as well as IL-6/IL-10 and IFN-γ/IL-10 ratios and further, to an early age at manifestation of SAR and poor sleep quality. These findings support a close relationship between acute allergic processes and affective states, with inflammatory cytokines, sleep, and age of manifestation as potentially relevant mediators.

Background The main goal of bone tissue engineering has been the generation of healthy bone in order to replace affected tissue. Therefore, optimized biomaterials are needed which allow the survival and growth of mesenchymal stem cells. Until now the key challenge in the clinical application of cell-based tissue engineering bone implants was poor diffusion of oxygen into the tissue, making functional blood vessel networks a necessity. With their ability to evolve into different cell types, to expand extensively in vitro, and to release paracrine soluble factors, bone marrow stromal cells (BMSC) are highly attractive for tissue engineering. During the last years hypoxia became a proven method to control proliferation, differentiation, and pluripotency of BMSC. Here we applied different methods to characterize metabolically conditioned media (MCM) in comparison to hypoxia conditioned media (HCM) and evaluated their ability to attract BMSC in 2-D migration assays. Methods BMSC and fibroblasts of human origin were isolated and cultivated to obtain HCM and MCM. Both media were characterized by angiogenesis arrays, cytokine arrays, and ELISA for selected factors. 2-D migration tests were performed with Corning Transwell®-96 permeable support chambers with porous polyester membranes with a pore size of 8.0 μm. Results Characterization of HCM and MCM revealed that the concentration of angiogenic factors was higher in MCM than in HCM. However, the chemoattractive capacity of MCM for BMSC was equivalent to that of HCM. HCM and MCM produced by human skin fibroblasts attracted human BMSC as efficiently as HCM and MCM produced by human BMSC. Conclusions HCM and MCM have a high chemoattractive capacity for BMSC. Both conditioned media harbor high concentrations of angiogenic factors which are important for angiogenesis and cell migration. Both chemoattracting conditioned media can also be derived from skin fibroblasts which can easily be obtained from patients in individualized therapy approaches.

Background Heterozygous point mutations in the GT splice donor consensus sequence of exon 11 of the PIK3R1 gene (coding for p85α, p55α, and p50α regulatory subunits of PI3K) lead to exon skipping and thereby to an aberrant protein that leaves PI3K hyperactivated. Several patients with this particular variant of PI3 kinase delta syndrome (APDS) suffering from sinopulmonary infections and lymphoproliferation have been described. Methods (Whole exome) sequencing, evaluation of cellular and clinical phenotypes. Results We here report a family with a new heterozygous mutation in this gene, a 9 bp deletion (c.1418_1425+1del) that, however, leads to the same skipping of exon 11. The clinical phenotypes of their members partly overlap features of patients of other reports. Conclusions We found a new mutation in PIK3R1 and show how broad the resulting clinical spectrum can be.

CASP1 variants result in reduced enzymatic activity of procaspase-1 and impaired IL-1β release. Despite this, affected individuals can develop systemic autoinflammatory disease. These seemingly contradictory observations have only partially been explained by increased NF-κB activation through prolonged interaction of variant procaspase-1 with RIP2. To identify further disease underlying pathomechanisms, we established an in vitro model using shRNA-directed knock-down of procaspase-1 followed by viral transduction of human monocytes (THP-1) with plasmids encoding for wild-type procaspase-1, disease-associated CASP1 variants (p.L265S, p.R240Q) or a missense mutation in the active center of procaspase-1 (p.C285A). THP1-derived macrophages carrying CASP1 variants exhibited mutation-specific molecular alterations. We here provide in vitro evidence for abnormal pyroptosome formation (p.C285A, p.240Q, p.L265S), impaired nuclear (pro)caspase-1 localization (p.L265S), reduced pro-inflammatory cell death (p.C285A) and changes in macrophage deformability that may contribute to disease pathophysiology of patients with CASP1 variants. This offers previously unknown molecular pathomechanisms in patients with systemic autoinflammatory disease.

Autoinflammatory diseases represent an expanding spectrum of diseases characterized by recurrent episodes of fever and systemic inflammation, due to disorders of innate immunity. Since the concept of autoinflammation was first affirmed in 1999 to define TNF-receptor associated periodic syndrome (TRAPS), many other monogenic autoinflammatory diseases have been identified. Uncontrolled secretion of IL-1β is responsible for the many of these syndromes, as also confirmed by the dramatic clinical response to IL-1 blockade. More recently, different mechanisms have been implicated in the pathophysiology of several monogenic autoinflammatory diseases, including cell stress, dysregulation in NF-κB signaling, ubiquitination, protein folding, type I interferon production and complement activation. In this chapter, we discuss IL-1β and other members of the IL-1 family and their inhibitors involved in monogenic autoinflammatory diseases, focusing on the mechanisms underlying their secretion in health and disease. Furthermore, we describe type I interferons and their role in autoinflammation.

Caspase-1 is a key player during the initiation of pro-inflammatory innate immune responses, activating pro-IL-1β in so-called inflammasomes. A subset of patients with recurrent febrile episodes and systemic inflammation of unknown origin harbor mutations in CASP1 encoding caspase-1. CASP1 variants result in reduced enzymatic activity of caspase-1 and impaired IL-1β secretion. The apparent paradox of reduced IL-1β secretion but systemic inflammation led to the hypothesis that CASP1 mutations may result in variable protein interaction clusters, thus activating alternative signaling pathways. To test this hypothesis, we established and characterized an in vitro system of transduced immortalized murine macrophages expressing either WT or enzymatically inactive (p.C284A) procaspase-1 fusion reporter proteins. Macrophages with variant p.C284A caspase-1 did not secrete IL-1β and exhibited reduced inflammatory cell death, referred to as pyroptosis. Caspase-1 and apoptosis-associated speck-like protein containing a CARD (ASC) formed cytosolic macromolecular complexes (so-called pyroptosomes) that were significantly increased in number and size in cells carrying the p.C284A caspase-1 variant compared with WT caspase-1. Furthermore, enzymatically inactive caspase-1 interacted with ASC longer and with increased intensity compared with WT caspase-1. Applying live cell imaging, we documented for the first time that pyroptosomes containing enzymatically inactive variant p.C284A caspase-1 spread during cell division. In conclusion, variant p.C284A caspase-1 stabilizes pyroptosome formation, potentially enhancing inflammation by two IL-1β-independent mechanisms: pyroptosomes convey an enhanced inflammatory stimulus through the recruitment of additional proteins (such as RIP2, receptor interacting protein kinase 2), which is further amplified through pyroptosome and cell division.

STING-associated vasculopathy with onset in infancy (SAVI) is an autoimmune disease caused by heterozygous gain of function mutations of STING (stimulator of interferon genes) that had initially been classified as a type I interferonopathy. We recently reported a genetically engineered mouse strain carrying a common SAVI-associated STING mutation. These STING N153S/WT mice reproduce key features of SAVI, including lung inflammation, loss of T cells in spleen and blood, splenomegaly and thymic hypoplasia. Here we show that αβ T lymphocytopenia is due to disrupted T cell development and is associated with impaired T cell activation and a relative increase in γδ T cell numbers. These alterations were not rescued by additional knockout of the type I IFN receptor (IFNAR1). Collectively, our findings consolidate the concept that constitutive STING signalling leads to a SCID-like phenotype in STING N153S/WT mice.

Introduction Bone marrow stromal cells (BMSC) are highly attractive for tissue engineering due to their ability to differentiate into different cell types, to expand extensively in vitro and to release paracrine soluble factors with a high regenerative potential. They were observed to migrate towards the sites of injury in response to chemotactic signals in vivo. During the last years hypoxia has become a proven method to control proliferation, differentiation and multipotency of BMSC. Conditioned medium from hypoxia-treated BMSC (Hypoxia-conditioned Medium; HCM) has been shown to have various favorable properties on tissue regeneration - such as on cell recruitment, wound healing, angiogenesis and revascularization. Due to this regenerative potential many studies attempt to further characterize HCM and its main functional components. In this study we used HCM generated from umbilical cord mesenchymal stem cells (UC-MSC) instead of BMSC, because GMP-verified methods were used to isolate and cultivate the cells and ensure their constant quality. UC-MSC have a high regenerative potential and are still immunologically naive and therefore highly unlikely to cause an immune reaction. In our article we took the first steps to closer investigate the role of umbilical cord MSC-derived HCM components, namely stromal cell-derived factor 1 (SDF-1α), interleukin 11 (IL-11) and soluble vascular cell adhesion molecule 1 (sVCAM-1). Results Our results show previously unknown roles of IL-11 and sVCAM-1 in the attraction of BMSC. The synergistic effect of the investigated protein mixture consisting of IL-11, sVCAM-1 and SDF-1α as well as those recombinant proteins alone revealed a significantly higher chemoattractive capacity towards human BMSC compared to normoxic control medium. Both, the protein mixtures and proteins alone as well as UC-HCM showed an angiogenic effect by promoting the formation of significantly longer tubule structures and higher amounts of junctions and tubules compared to normoxic control medium. Conclusions By showing the prominent upregulation of IL-11, sVCAM-1 and SDF-1α under hypoxic conditions compared to normoxic control and revealing their crucial role in migration of human BMSC we took a further step forward in characterization of the chemoattractive components of HCM.

Subcellular localization studies and life cell imaging approaches usually benefit from fusion-reporter proteins, such as enhanced green fluorescent protein (EGFP) and mCherry to the proteins of interest. However, such manipulations have several risks, including protein misfolding, altered protein shuttling, or functional impairment when compared to the wild-type proteins. Here, we demonstrate altered subcellular distribution and function of the pro-inflammatory enzyme procaspase-1 as a result of fusion with the reporter protein mCherry. Our observations are of central importance to further investigations of subcellular behavior and possible protein-protein interactions of naturally occurring genetic variants of human procaspase-1 which have recently been linked to autoinflammatory disorders.

Osteoporosis induction in a sheep model by steroid administration combined with ovariectomy recapitulates decreased bone formation and substandard matrix mineralization in patients. Recently, the role of osteocytes has been frequently addressed, with focus on their role in osteoclastogenesis. However, the quantification of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) signaling in osteocytes was not studied in sheep. The current study reproduced the sheep model of osteoporosis to study the RANKL/OPG ratio correlation to the method of osteoporosis induction. We investigated the induction of osteoporosis after 8 months using 31 female merino land sheep divided into four groups: control, ovariectomy, ovariectomy with dietary limitation, and ovariectomy with dietary limitation and steroid injection. In accordance to previous reports, the present study showed trabecular thinning, higher numbers of apoptotic osteocytes, and imbalanced metabolism, leading to defective mineralization. The global RANKL/OPG ratio in the spine after 8 months of steroid and dietary treatment was not different from that of the control. Interestingly, assessment of the osteocyte-specific RANKL/OPG ratio showed that the steroid-induced osteoporosis in its late progressive phase stimulates RANKL expression in osteocytes. Sclerostin is suggested to induce RANKL expression in osteocytes. The findings of this study can contribute to further explain the success of sclerostin antibodies in treating osteoporotic patients despite increased osteocyte-expressed RANKL.

The proinflammatory enzyme caspase-1 plays an important role in the innate immune system and is involved in a variety of inflammatory conditions. Rare naturally occurring human variants of the caspase-1 gene (CASP1) lead to different protein expression and structure and to decreased or absent enzymatic activity. Paradoxically, a significant number of patients with such variants suffer from febrile episodes despite decreased IL-1β production and secretion. In this study, we investigate how variant (pro)caspase-1 can possibly contribute to inflammation. In a transfection model, such variant procaspase-1 binds receptor interacting protein kinase 2 (RIP2) via Caspase activation and recruitment domain (CARD)/CARD interaction and thereby activates NF-κB, whereas wild-type procaspase-1 reduces intracellular RIP2 levels by enzymatic cleavage and release into the supernatant. We approach the protein interactions by coimmunoprecipitation and confocal microscopy and show that NF-κB activation is inhibited by anti–RIP2-short hairpin RNA and by the expression of a RIP2 CARD-only protein. In conclusion, variant procaspase-1 binds RIP2 and thereby activates NF-κB. This pathway could possibly contribute to proinflammatory signaling.

Inflammasomes are multiprotein complexes that serve as activating platforms for the enzyme caspase-1 in response to various danger signals. Active caspase-1 can cleave the precursors of the pro-inflammatory cytokines IL-1β and IL-18 and thereby activate them. Deregulation of this cascade caused by mutations in genes coding for inflammasomal components and their interaction partners can lead to severe disease. This review summarizes the contribution of deregulated inflammasomes to the field of autoinflammatory syndromes. In addition, it gives insight into currently available mouse models that are used to study and characterize the role of the inflammasome components in the pathophysiology of these diseases.

Chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory bone disorder of unknown origin. We previously demonstrated that monocytes from CRMO patients fail to express the immune-modulatory cytokine interleukin-10 (IL-10) in a chromatin dependent manner. Here, we demonstrate that attenuated extracellular-signal regulated kinase (ERK)1 and 2 signaling in response to TLR4 activation results in failure to induce IL-10 expression in monocytes from CRMO patients. Attenuated ERK1/2 activation results in 1) reduced levels of Sp-1, a transcription factor that induces IL-10 expression in monocytes, and 2) impaired H3S10 phosphorylation of the IL10 promoter, an activating epigenetic mark. The pro-inflammatory cytokines tumor necrosis factor (TNF)α and IL-6 are not negatively affected, resulting in an imbalance towards pro-inflammatory cytokines. Thus, impaired ERK1/2 signaling with subsequently reduced Sp-1 expression and H3S10 phosphorylation of the IL10 promoter may centrally contribute to the pathophysiology of CRMO.

The IL-10 cytokine family has nine members, four of which are located in the IL10 cluster on chromosome 1q32. These cytokines are the immune regulatory cytokine IL-10 itself, and the IL-20 subfamily members IL-19, IL-20, and IL-24. IL-10 instructs innate and adaptive immune responses and limits pro-inflammatory responses in order to prevent tissue damage. The IL-20 subfamily members are involved in host defense mechanisms, particularly from epithelial cells and seem essential for tissue integrity. Dysregulation of IL-10 family cytokines results in inflammation and autoimmune disease. Here, we discuss cellular source, gene regulation, and receptor complexes of cytokines in the IL10 cluster and their contribution to autoimmune disease and tissue damage.

Chronic non-bacterial osteomyelitis (CNO) is an auto-inflammatory disorder that affects the skeletal system. Interleukin (IL-)10 is an immune-modulatory cytokine that controls inflammation, and limits inflammatory cytokine responses. Dysregulation of IL-10 expression has been shown to result in autoimmune and infectious diseases. We investigated IL-10 expression by monocytic cells from CNO patients and controls. In response to stimulation with LPS, IL-10 expression from CNO monocytes was reduced (p

Caspase-1, belonging to the family of cystein proteases, is well-known for its involvement in IL-1β and IL-18 maturation. Although, caspase-1 is one of the best described caspase-family-members, open questions concerning autoinflammatory diseases, caused by procaspase-1 variants (e.g. ICE-Fever), remain. ICE-Fever patients suffer from chronic febrile episodes, although procaspase-1 variants show reduced enzymatic activity leading to limited IL-1β secretion. The paradox of reduced IL-1β secretion but increased inflammation led to the hypothesis, that CASP1-variants enhance alternative signaling pathways. However, the role of procaspase-1 variants and their interaction partners during IL-1β maturation or NF-κB activation is still poorly understood.

Caspase-1 is a unique cysteine protease playing central roles in innate immunity. Pathogens, stress, and damage signals induce activation of caspase-1, typically mediated by proximity-induced autoproteolysis in multimeric protein complexes called the inflammasome. Active caspase-1 induces secretion of pro-inflammatory cytokines and mediates pyroptosis, a programmed pro-inflammatory cell death, thereby initiating an immune response finally leading to pathogen clearance. Excessive activation of caspase-1 is the underlying cause for rare diseases such as periodic fever syndromes, and more common disorders, including atherosclerosis, type 2 diabetes, and gout. Beside these well-known pro-inflammatory functions, active caspase-1 also has anti-inflammatory and protective functions contributing to cell survival, reduced inflammatory cytokine signaling, and improved outcomes in mouse models of burn injury or trauma and shock. Furthermore, naturally occurring procaspase-1 variants with reduced or abrogated enzymatic activity mediate enhanced inflammatory signaling and have been associated to autoinflammatory symptoms. Here, we review functions of caspase-1 focusing on anti-inflammatory signaling pathways and discuss the role of enzymatically inactive caspase-1 as disease-promoting factors in autoinflammatory diseases. Moreover, we illustrate differential requirements for autoproteolysis and enzymatic activity in caspase-1 functions.

We have recently reported that the RD114-pseudotyped MFGS-gp91phox vector achieves unprecedented levels of correction of the NADPH-oxidase gp91phox (approved gene symbol CYBB) defect in CD34(+) cells from patients with X-linked chronic granulomatous disease in the NOD/SCID mouse model. Considering clinical use of this vector, we transplanted autologous mobilized peripheral blood CD34(+) progenitor cells, transduced with the RD114-MFGS-gp91phox vector, into two healthy rhesus macaques following nonmyeloablative conditioning. The moderately high levels of in vivo marking seen in the first months following transduction decreased and stabilized at about 8 months posttransplant. Marking for both healthy animals after 15 months was 0.3 to 1.3 vector copies per 100 cells in lymphocytes, neutrophils, and monocytes. Vector insertion analyses performed by linear amplification-mediated PCR and sequencing identified 32 and 45 separate insertion sites in the animals. Identical insertion sites were found in myeloid cells and lymphocytes, demonstrating the successful transduction of lymphomyeloid progenitors. Some inserts landed in the vicinity of genes controlling cell cycle and proliferation. Statistical analyses of insertion sites 1 year posttransplant suggest a high diversity of insertion sites despite low marking.

Chronic granulomatous disease (CGD) is caused by a defect in both the host's defenses and its regulation of inflammation normally provided by phagocytes and other leukocytes. As in the case described here, it is not uncommon that CGD patients are diagnosed late, only after organ-damaging manifestations have already progressed. In this patient, we found that CGD arose due to a splice-supporting mutation in the last position of a cryptic exon towards the middle of intron 6 of the CYBB (gp91-phox) gene. The mutation led to the insertion of 56 bp into most of the CYBB mRNA of leukocytes causing a frame shift and a premature stop codon. The normal cryptic exon was also found to be mildly active in some tissues other than leukocytes in healthy donors, to be conserved in many primates, and to a lesser degree in other mammals. Some sequence similarity suggests that the cryptic exon may have originated from a mammalian interspersed repetitive (MIR) element. Taken together, we clarify an unusual disease-causing mutation, indicate its evolutionary background and emphasize the importance of a timely diagnosis of CGD.

P-glycoprotein (Pgp) is a well-defined ATP-binding cassette (ABC) protein and a close relative of cystic fibrosis transmembrane conductance regulator (CFTR), whose dysfunction causes cystic fibrosis (CF). It is postulated that Pgp can complement deficient CFTR functions because of structural and functional homologies. One of the most relevant functions appears to be the regulation of ATP release, which influences mucociliary clearance in respiratory epithelia by nucleotide receptor stimulation. However, mechanisms involved in ATP secretion remain a controversial issue. In the present study, CF epithelial cells (sigmaCFTE29o) were transduced with the retroviral vector MP1m encoding Pgp, and thus, a stable Pgp-overexpressing CF cell line (sigmaCFTE29oPgp) was established and used for studies of hypothesized CFTR complementation. In addition, overexpression of native Pgp in sigmaCFTE29o could also be achieved by long-term treatment with colchicine, a drug, which may be of great interest in CF therapy. We confirmed that overexpression of Pgp causes a significant increase in cellular ATP release, which could even be enhanced by stimulation with hypoosmolar medium. A potential clinical benefit is discussed.

Autoimmune lymphoproliferative syndrome type Ia (ALPS Ia) is caused by mutations in the CD95/APO1/FAS (TN-FRSF6) gene, which lead to a defective CD95 ligand (CD95L)–induced apoptosis. Soluble CD95 (sCD95) has been suggested to play an important role in the pathogenesis of diverse autoimmune and malignant diseases by antagonizing CD95L. Here we evaluate a family with 4 of its 5 members harboring an ex-6–3C→G mutation that affects the splice cis regulatory region (cctacag/ex-6→cctagag/ex-6) of the CD95 gene. The mutation causes skipping of exon-6, which encodes the transmembrane region of CD95, and thereby leads to an excessive production of sCD95 in all 4 affected individuals. The mutation is associated with a low penetrance of disease phenotype and caused mild and transient ALPS in one male patient whereas all other family members are completely healthy. In all family members with the mutation we found that the cell surface expression of CD95 was low and the activated T cells were resistant to CD95-induced apoptosis. Unexpectedly, excessive production or addition of sCD95 had no effect on the CD95-induced apoptosis in diverse cells. In contrast, increasing the surface expression of CD95 was able to correct the defect in apoptosis. Thus we conclude that the ALPS in the one male patient was caused by haploinsufficiency of membrane CD95 expression. Our data challenge the hypothesis that sCD95 causes autoimmunity.

Dominant mutations in the CIAS1 gene cause a spectrum of autoinflammatory diseases such as familial cold autoinflammatory syndrome, FCAS, which is characterized by episodes of urticaria, arthralgia, fever and conjunctivitis after generalized exposure to cold. We here describe patients of two German families with the 592G-->A, V198M mutation, which has been described to induce FCAS before. However, in our patients the clinical phenotype was very different from this disease. They never had urticaria, cold induced fever or conjunctivitis; instead the following symptoms occurred: Very regular periodic fever, irregular severe febrile episodes, relatively mild arthralgia, dry cough, cardiomyopathy, nephropathy and euthyroid thyroiditis all being reversible. We conclude that the clinical phenotype associated with mutations in the CIAS1 gene is much broader than assumed before.

Reactive oxygen intermediates (ROI) generated in the respiratory burst reaction are crucial for the killing of bacteria and fungi in phagocytes. The key enzyme for the respiratory burst reaction is the NADPH oxidase. Reactive oxygen intermediates have additionally been proposed to be of general importance for the expression of FAS and soluble FAS ligand (sFASL) and the subsequent induction of apoptosis. This conclusion has been drawn from the observation that neutrophils with an inborn lack of the NADPH oxidase as well as cell lines and monocytes with artificially blocked NADPH oxidase exhibit impaired apoptosis. Being one of the few centers caring for patients with chronic granulomatous disease (CGD) who exhibit an inborn lack of NADPH oxidase, we had the unique opportunity to determine the role of the NADPH oxidase for apoptosis in monocytes with otherwise unmanipulated cells of these patients (CGD monocytes). We compared the expression of FAS on monocytes and the concentration of sFASL in the supernatant between CGD monocytes and healthy donors undergoing spontaneous apoptosis. Neither the expression of FAS nor the concentration of sFASL was decreased in CGD monocytes. We further compared spontaneous apoptosis and apoptosis occurring after the phagocytosis of Staphylococcus aureus in CGD monocytes to monocytes of healthy controls. In these experiments we could not determine any significant impairment of apoptosis in CGD monocytes. Our data indicate for the first time that in an unmanipulated human model a functional NADPH oxidase is not crucial for the apoptosis of monocytes and disprove a general role of ROI for the induction of apoptosis in phagocytes.

The collection of sputum for microbiological examination in young cystic fibrosis patients can be very difficult. However, a knowledge of bacterial flora colonizing the patient's airways is of paramount importance for proper antimicrobial therapy. It is also known that cystic fibrosis patients colonized by Pseudomonas species have a poorer prognosis than Pseudomonas-negative patients. Noninvasive ways of diagnosing airway inflammation that require only minimal cooperation of the patient might yield new possibilities for early detection of airway colonisation. The breath condensate method as a noninvasive diagnostic technique seems especially appropriate for use in children. Therefore, the aim of this study was to evaluate whether the breath condensate method could be used for detection of Pseudomonas species in children with cystic fibrosis. In total, 32 breath condensate and seven sputum samples were obtained from 13 cystic fibrosis patients with Pseudomonas aeruginosa- or Burkholderia cepacia-positive sputum culture (20 samples were obtained during forced expiration). PCR for combined detection of Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Burkholderia cepacia was performed. PCR results of all breath condensate samples were negative for Pseudomonas aeruginosa, Stenotrophomonas maltophilia, or Burkholderia cepacia, while all sputum sample results were positive. A minimum DNA quantity of 10 fg could be detected in dilution series of the positive control group. We conclude that the breath condensate method cannot be used as a tool for detection of Pseudomonas species.

Lymphomas were studied in kindreds with autoimmune lymphoproliferative syndrome (ALPS; Canale-Smith syndrome), a disorder of lymphocyte homeostasis usually associated with germline Fas mutations. Fas (CD95/APO-1) is a cell surface receptor that initiates programmed cell death, or apoptosis, of activated lymphocytes. Lymphoma phenotype was determined by immunohistochemistry, frequency of CD3(+)CD4(-)CD8(-) T-cell-receptor alpha/beta cells by flow cytometry, nucleotide sequences of the gene encoding Fas (APT1, TNFRSF6), and the percentage of lymphocytes undergoing apoptosis in vitro. Of 223 members of 39 families, 130 individuals possessed heterozygous germline Fas mutations. Eleven B-cell and T-cell lymphomas of diverse types developed in 10 individuals with mutations in 8 families, up to 48 years after lymphoproliferation was first documented. Their risk of non-Hodgkin and Hodgkin lymphomas, respectively, was 14 and 51 times greater than expected (each P

We describe the history of a girl with interferon-gamma-receptor (IFNgammaR1) deficiency and studies performed to identify the molecular and clinical characteristics of this recently discovered disorder. This is the first report of a child from Northern Europe with IFNgammaR1 deficiency. The patient, now 7 years old, first presented with swelling and reddening at the Bacille Calmette-Guerin (BCG) vaccination site, swelling of lymph nodes, hepatomegaly, and an unusually severe varicella rash at the age of 4 months. At that time, she was diagnosed with BCG histiocytosis without typical granuloma formation and was treated with antituberculous agents. During the clinical course of her illness, several different types of atypical mycobacteria and (for the first time in an IFNgammaR1-deficient patient) Listeria monocytogenes were detected. Flow cytometric analysis showed that the patient's monocytes could not bind a monoclonal antibody specific for the IFNgamma-receptor. Our analysis of mRNA derived from the alpha-chain (IFNgammaR1) gene of this receptor revealed deletions of 173 bp and 4 bp in cDNA sequences originating from individual alleles. The 173 bp deletion was located between nucleotide positions 200 and 372, exactly matching those of exon 3, and the 4 bp deletion was located between nucleotide positions 561 and 564 of the coding region of the cDNA. Analysis of genomic DNA revealed the presence of a G to T transition at the 5'end of the splice consensus sequence of intron 3, which explains the absence of exon 3. The other allele carried the 4-base-pair deletion (ACTC) at nucleotide positions 15-18 of exon 5. Twelve months after an allo\geneic bone marrow transplantation, the patient had clinically improved.

Dear Editor, We would like to add some new results to our recent publication about Alu repeat-induced autosomal recessive chronic granulomatous disease (CGD) [1]. CGD patients are susceptible to severe infections with certain opportunistic bacteria and fungi and also often suffer from granulomatous inflammation even in the absence of infection. The disorder is caused by genetically determined defects in components of the phagocyte multienzyme complex NADPH oxidase. Recently, we characterized several patients with autosomal recessive p67-phox-deficient CGD evoked by an Alu repeat-induced deletion within the NCF2 gene [1]. The NCF1 gene encoding p47-phox also contains several Alu repeats that may contribute to recombination events with one of the two highly homologous unprocessed NCF1 pseudogenes, thereby causing p47-phox-deficient CGD [2, 3]. In this letter, we show for the first time that autosomal recessive p47-phox-deficient CGD can also be caused by an Alu repeat-induced deletion within the NCF1 gene. We found two CGD patients of different families with the same mutation c.154−283_451+821del2858 in NCF1 [4] and, thus, a deletion of exons 3–5 in their p47-phox cDNA. In one of the patients, this deletion was present in homozygous form; in the other, in heterozygous form together with a complete NCF1 deletion. The two families are most likely unrelated, and only one of the patients has consanguineous parents. One family is of Polish, and the other is of German origin. Furthermore, a short tandem repeat (STR, hg19_chr7, 7201262–7201312) located 2 Mb from the NCF1 gene has nine repeats in one and ten repeats in the other family, and an STR close to the NCF1 gene (hg19_chr7, 73943032–73943364) differs by one repeat on the allele with the complete deletion of NCF1 and by two repeats on the allele with the deletion of exons 3–5 between the two families (data not shown). Surprisingly, we nevertheless found exactly the same genomic sequence in both patients (Fig. 1, marked in gray). The Alu repeat located downstream of exon 2 crosses over to the Alu repeat upstream of exon 6, thereby deleting the 2,858-bp DNA stretch comprising exons 3–5. The putative crossover region is marked in dark gray, and possible gene conversions are indicated by bold arrows. Such gene conversions are frequently associated with Alu-repeat induced crossovers (see also figure legend). Specific analysis of the cDNA with the three-exon deletion (thus avoiding pseudogene cDNA) revealed that all SNPs in exonic regions [2] were of NCF1 wild type and not of pseudogene origin (data not shown). Therefore, we found no hint of a crossover of the wild type with one of the NCF1 pseudogenes, even though we cannot completely rule out this possibility. In conclusion, Alu repeats can cause deletions not only in the NCF2 but also in the NCF1 gene, resulting in autosomal recessive CGD in both instances. Breakpoints and gene conversion hot spots within the Alu repeats of these genes Stefan Winkler, Karin van Leeuwen, Dirk Roos, and Joachim Roesler contributed equally.

Annexin A2 (ANXA2) promotes myeloma cell growth, reduces apoptosis in myeloma cell lines, and increases osteoclast formation. ANXA2 has been described in small cohorts of samples as expressed by myeloma cells and cells of the BM microenvironment. To investigate its clinical role, we assessed 1148 samples including independent cohorts of 332 and 701 CD138-purified myeloma cell samples from previously untreated patients together with clinical prognostic factors, chromosomal aberrations, and gene expression–based high-risk scores, along with expression of ANXA2 in whole BM samples, stromal cells, osteoblasts, osteoclasts, and BM sera. ANXA2 is expressed in all normal and malignant plasma cell samples. Higher ANXA2 expression in myeloma cells is associated with significantly inferior event-free and overall survival independently of conventional prognostic factors and is associated with gene expression–determined high risk and high proliferation. Within the BM, all cell populations, including osteoblasts, osteoclasts, and stromal cells, express ANXA2. ANXA2 expression is increased significantly in myelomatous versus normal BM serum. ANXA2 exemplifies an interesting class of targetable bone-remodeling factors expressed by normal and malignant plasma cells and the BM microenvironment that have a significant impact on survival of myeloma patients.

Caspase-1 (Interleukin-1 Converting Enzyme, ICE) is a proinflammatory enzyme that plays pivotal roles in innate immunity and many inflammatory conditions such as periodic fever syndromes and gout. In- flammation is often mediated by enzymatic activation of interleukin (IL)-1β and IL-18. We detected seven naturally occurring human CASP1 variants with different effects on protein structure, expression, and enzymatic activity. Most mutations destabilized the caspase-1 dimer interface as revealed by crystal structure analysis and homology modeling followed by molecular dynamics simulations. All variants demonstrated decreased or absent enzymatic and IL-1β releasing activity in vitro, in a cell transfection model, and as low as 25% of normal ex vivo in a whole blood assay of samples taken from subjects with variant CASP1, a subset of whom suffered from unclassified autoinflammation. We conclude that decreased enzymatic activity of caspase-1 is compatible with normal life and does not prevent moderate and severe autoinflammation. Fil: Luksch, Hella. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; Alemania Fil: Romanowski, Michael J.. Sunesis Pharmaceuticals. Department of Structural Biology; Estados Unidos Fil: Chara, Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física de Líquidos y Sistemas Biológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física de Líquidos y Sistemas Biológicos; Argentina. Technische Universitat Dresden. Center for Information Services and High-Performance Computing; Alemania Fil: Tuengler, Victoria. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Caffarena, Ernesto Raúl. Escola Nacional de Saude Publica Sergio Arouca. Fundación Oswaldo Cruz; Brasil Fil: Heymann, Michael C.. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; Alemania Fil: Lohse, Peter. Ludwig-Maximilians-Universit; Alemania Fil: Aksentijevich, Ivona. Inflammatory Disease Section; Estados Unidos Fil: Remmers, Elaine F.. Inflammatory Disease Section; Estados Unidos Fil: Flecks, Silvana. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; Alemania Fil: Quoos, Nadine. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; Alemania Fil: Gramatté, Johannes. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; Alemania Fil: Petzold, Cathleen. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; Alemania Fil: Hofmann, Sigrun R.. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; Alemania Fil: Winkler, Stefan. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; Alemania Fil: Pessler, Frank. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; Alemania. Helmholtz Centre for Infection Research; Alemania Fil: Kallinich, Tilmann. Universität zu Berlin; Alemania Fil: Ganser, Gerd. Sendenhorst. St. Josef-Stift; Alemania Fil: Nimtz-Talaska, Antje. Kinderrheumatologie; Alemania Fil: Baumann, Ulrich. Hannover Medical School; Alemania Fil: Runde, Volker. Wilhelm-Anton-Hospital; Alemania Fil: Grimbacher, Bodo. University Hospital Freiburg; Alemania Fil: Birmelin, Jennifer. University Hospital Freiburg; Alemania Fil: Gahr, Manfred. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; Alemania Fil: Roesler, Joachim. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; Alemania Fil: Rösen-Wolff, Angela. University Hospital Carl Gustav Carus. Department of Pediatrics. Dresden; Alemania

Two brothers in their fifties presented with a medical history of suspected fungal allergy, allergic bronchopulmonary aspergillosis, alveolitis, and invasive aspergillosis and pulmonary fistula, respectively. Eventually, after a delay of 50 years, chronic granulomatous disease (CGD) was diagnosed in the index patient. We found a new splice mutation in the NCF2 (p67-phox) gene, c.1000 + 2T → G, that led to several splice products one of which lacked exons 11 and 12. This deletion was in frame and allowed for remarkable residual NADPH oxidase activity as determined by transduction experiments using a retroviral vector. We conclude that p67-phox which lacks the 34 amino acids encoded by the two exons can still exert considerable functional activity. This activity can partially explain the long-term survival of the patients without adequate diagnosis and treatment, but could not prevent progressing lung damage.

A comparison of the pathogenicity in mice of the recombinant herpes simplex virus type 1 (HSV-1) strain HSV-1-M- LacZ, in which the UL56 gene has been deleted, was made with its parental strain F, following infection in different mouse strains. The polymerase chain reaction (PCR) technique was used to study the migration of virus DNA in the mouse model. Tissues from adult mice infected intraperitoneally (IP) with one of three HSV-1 strains (F, HFEM or HSV-1-LacZ) were examined for the presence of viral DNA. DNA of the pathogenic strain F was detected in the adrenal glands, spinal cord, brain, liver and pancreas. DNA of HSV-1-M-LacZ was detected in the same tissues. However, DNA of the apathogenic strain HFEM was detected transiently (on days 2 and 3 p.i., but not days 1, 5 or 7), only in the adrenal glands and no viral DNA was detected in any of the other tissues. HSV-1 pathogenic strains injected intraperitoneally into newborn mice (7 days old) killed most of the mice. In the surviving mice viral DNA of the three virus strains was found in peritoneal exudate cells (PEC), adrenal glands, spinal cord, liver and spleen. It was found that HSV-1-M-LacZ, which lacks the UL56 gene, resembled in pathogenicity to the newborn mice the pathogenic HSV-1 strains F and KOS. The PCR technique was used to trace viral DNA in tissues of the mice which survived HSV-1 infection at 7 weeks of age. Only HSV-1 (KOS) DNA was detected in the pancreas. The brains of these mice did not contain viral DNA. It is suggested that HSV-1 DNA may reside in surviving HSV-1- infected newborn mice in a “latent” state in nonneural tissues.

The repetitive DNA sequences of the genome of Molluscum contagiosum virus type 1 (MCV-1) have been localized within the terminal regions of the viral genome corresponding to the BamHI MCV-1 DNA fragments B (18 kbp; 0 to 0.095 map units (m.u.)) and E (10.5 kbp; 0.944 to 1 m.u.). The fine mapping of these particular regions of the genome of MCV-1 revealed that the boundaries of the terminal repetitive DNA sequences of the viral genome are located within the DNA sequences of the HindIII MCV-1 DNA fragments K (3.8 kbp; 0.014 to 0.036 m.u.) and J1 (4.1 kbp; 0.962 to 0.985 m.u.). The exact position of the boundary of the repetitive DNA sequences was determined by DNA nucleotide sequencing. The HindIII DNA fragments K and J1 compose 3859 and 4107 bp, respectively. The DNA sequences of HindIII MCV-1 DNA fragment K possess repetitive DNA sequences between the nucleotide positions 1 and 1675 which are homologous to the inverted and complementary DNA sequences of the HindIII MCV-1 DNA fragment J1 between the nucleotide positions 2437 and 4107 (1670 bp). The degree of DNA sequence homology detected between the repetitive DNA sequences in the HindIII DNA fragments K and J1 of the viral genome was found to be 98%. The number of open reading frames (ORFs) detected by the analysis of the DNA sequences of the HindIII MCV-1 DNA fragments K and J1 was found to be 14 (70 to 219 amino acid residues) and 11 (70 to 365 amino acid residues), respectively.

Due to their great potential, mesenchymal stem cells (MSC) are of special interest for regenerative therapies. One of the most investigated areas in this field is the regeneration of bone tissue using tissue engineering strategies. This chapter focuses on osteogenic differentiation and cultivation of human MSC in vitro and their utilisation for tissue engineering of bone tissue, discussing a number of protocols to induce and influence the osteogenic differentiation of MSC into the osteoblastic lineage in vitro.

The origins of DNA replication of the genome (209 kbp) of Chilo iridescent virus (CIV), which is circularly permuted and terminally redundant, were identified. The defined genomic library of CIV, which represents 100% of DNA sequences of the viral genome (e.g., all 32 EcoRI CIV DNA fragments), was used for transfection of Choristoneura fumiferana insect cell cultures (CF-124) that were previously infected with CIV. The plasmid rescue experiments were carried out to select those recombinant plasmids that were amplified during viral replication in CIV-infected cell cultures. It was found that six recombinant plasmids harboring the EcoRI DNA fragments C [13.5 kbp, 0.909-0.974 map units (m.u.)], H (9.8 kbp, 0.535-0.582 m.u.), M (7.25 kbp, 0.310-0.345 m.u.), O (6.5 kbp, 0.196-0.228 m.u.), Q (5.9 kbp, 0.603-0.631 m.u.), and Y (2.0 kbp, 0.381-0.391 m.u.) were able to be amplified under the conditions used. This indicates that the CIV genome possesses six DNA replication origins. Subclones of the EcoRI CIV DNA fragments C and H were screened under the same conditions. It was found that DNA sequences within the EcoRI DNA fragments C and H at the genome coordinates 0.924-0.930 and 0.535-0.548, respectively, contain origins of viral DNA replication. The DNA nucleotide sequences of the EcoRI CIV DNA fragment Y (1986 bp) were determined for identifying the DNA sequence of the corresponding origin of DNA replication. The computer-aided analysis revealed the presence of a 15-mer inverted repeat at nucleotide positions 661-675 and 677-691 (661-TAAATTTAATGAGAA-G-TTCTCATTAAATTTA-692). The analysis of the DNA sequence of the EcoRI DNA fragment H corresponding to the particular region at the genome coordinates 0.535-0.548 (1) showed that this region contains a 16-mer inverted repeat at the nucleotide positions 1315 and 1332 (1315-TAAATTTTAATGGTTA-A-TAACCATTAAAATTTA-1347), which is very similar to the inverted repetition found within the EcoRI DNA fragment Y. The successful recognition and amplification of the single-stranded synthetic DNA sequences of both strands of CIV-ori-Y (nucleotide position 661-691) using phage M13 system in CIV-infected cells is strong evidence that the CIV-ori-Y is bidirectionally active, and this DNA sequence is considered to be the origin of DNA replication within the EcoRI CIV DNA fragment Y.

The BamHI-B DNA fragment of herpes simplex virus type-1 (HSV-1) is associated with intraperitoneal pathogenicity. Among the recently mapped RNA transcripts from this fragment (15), one was reported to be associated with latency. To relate the RNA transcripts to virus pathogenicity, the in vitro-transcribed RNAs from BamHI-B fragments of three HSV-1 strains--F (pathogenic), R19, and HFEM (apathogenic), were studied by in vitro translation. When the BamHI-HpaI (0.738-0.755 map units) DNA fragment from HSV-1 strain F was transcribed rightward and translated, three proteins of 70, 63, and 51 kD were detected. The 63 kD protein resembles in size and orientation the protein encoded by the ICP-27 (IE-2) gene (0.740-0.749 mu). The 51 kD polypeptide is assumed to be a prematurely terminated form of this protein. No proteins were obtained from RNA transcribed in the opposite direction. The SalI-NcoI (0.746-0.761 mu) fragment of the three HSV-1 strains yielded two proteins of 25 and approximately 15 kD when transcribed rightward and a 35 kD polypeptide from RNA transcribed in the opposite direction. As a result of the genomic deletion in HFEM, it was possible to obtain the 35 kD protein from the SalI-SalI DNA fragment (0.746-0.761 mu) as well. In vitro transcription and translation of the PstI-SalI (0.778-0.790 mu) DNA fragment (the right-hand side of HpaI-P) did not result in protein synthesis. The possibility that the UL56 gene is connected with the intraperitoneal pathogenicity of HSV-1 is discussed.

Background: The flow cytometric dihydrorhodamine 123 (DHR) assay is used as a screening test for chronic granulomatous disease (CGD), but complete myeloperoxidase (MPO) deficiency can also lead to a strongly decreased DHR signal. Our aim was to devise simple laboratory methods to differentiate MPO deficiency (false positive for CGD) and NADPH oxidase abnormalities (true CGD).Methods: We measured NADPH-oxidase and MPO activity in neutrophils from MPO-deficient patients, CGD patients, NADPH-oxidase–transfected K562 cells and cells with inhibited and substituted MPO.Results: Eosinophils from MPO-deficient individuals retain eosinophilic peroxidase and therefore generate a normal DHR signal. The addition of recombinant human MPO enhances the DHR signal when simply added to a suspension of MPO-deficient cells but not when added to NADPH-oxidase–deficient (CGD) cells. Lucigenin-enhanced chemiluminescence (LCL) is increased in neutrophils from MPO-deficient patients, whereas neutrophils from patients with CGD show a decreased response.Conclusions: A false-positive result caused by MPO deficiency can be easily ascertained because, unlike cells from a CGD patient, cells from MPO-deficient patients (a) contain functionally normal eosinophils, (b) show a significant enhancement of the DHR signal following addition of rhMPO, and (c) generate a strong LCL signal.

Caspase-1 activates proIL-1β and proIL-18 and plays a fundamental role in innate immunity. This pro-inflammatory immune response is required for the initiation of pathogen clearance. Caspase-1 itself is activated in so-called “inflammasomes” which are assembled in response to distinct pathogen associated molecular patterns (PAMP) or danger associated molecular patterns (DAMP). Most studies analyzing the inflammasome/caspase-1 activity of patients ex vivo use PBMC-based assays. Beside monocytes and macrophages, caspase-1 is also activated in PMNs representing the major leukocyte subset in peripheral blood. Thus, analyzing purified PBMCs seems to be highly artificial by excluding caspase-1 activity in PMNs and ignoring cellular interactions. Furthermore, PBMC purification requires large amounts of blood, thereby rendering the assay impractical for the use in small children or neonates.

The transcription factor signal transducer and activator of transcription-1 (STAT1) plays a key role in immunity against mycobacterial and viral infections. Here, we characterize three human STAT1 germline alleles from otherwise healthy patients with mycobacterial disease. The previously reported L706S, like the novel Q463H and E320Q alleles, are intrinsically deleterious for both interferon gamma (IFNG)–induced gamma-activating factor–mediated immunity and interferon alpha (IFNA)–induced interferon-stimulated genes factor 3–mediated immunity, as shown in STAT1-deficient cells transfected with the corresponding alleles. Their phenotypic effects are however mediated by different molecular mechanisms, L706S affecting STAT1 phosphorylation and Q463H and E320Q affecting STAT1 DNA-binding activity. Heterozygous patients display specifically impaired IFNG-induced gamma-activating factor–mediated immunity, resulting in susceptibility to mycobacteria. Indeed, IFNA-induced interferon-stimulated genes factor 3–mediated immunity is not affected, and these patients are not particularly susceptible to viral disease, unlike patients homozygous for other, equally deleterious STAT1 mutations recessive for both phenotypes. The three STAT1 alleles are therefore dominant for IFNG-mediated antimycobacterial immunity but recessive for IFNA-mediated antiviral immunity at the cellular and clinical levels. These STAT1 alleles define two forms of dominant STAT1 deficiency, depending on whether the mutations impair STAT1 phosphorylation or DNA binding., Synopsis Mendelian susceptibility to mycobacterial disease is a rare syndrome. It is defined by the occurrence of severe disease caused by low virulence mycobacteria in otherwise healthy individuals, in whom antiviral immune response is not affected. Eleven known genetic defects, affecting five genes, have been involved in this type of deficient response to infection, involving immune-mediator molecules IL12 and interferon gamma: IL12B, IL12RB1, IFNGR1, IFNGR2, and STAT1. The signal transducer and activator of transcription-1 (STAT1) amino acid change L706S was previously shown to cause disease by impairing STAT1 phosphorylation. Here, we report two new STAT1 mutations that impair STAT1 DNA-binding activity. We show, by functional analysis of the three STAT1 mutant alleles, that they are intrinsically deleterious for both interferon gamma–induced antimycobacterial immunity, which is mediated through gamma-activated factor and for interferon alpha–induced antiviral immunity, which is mediated through interferon-stimulated genes factor 3. Interestingly, the three alleles are dominant for interferon gamma–induced gamma-activated factor–mediated antimycobacterial immunity, but recessive for interferon alpha–induced interferon-stimulated genes factor 3–mediated antiviral immunity at the cellular and clinical levels. These two new STAT1 alleles, which affect the binding of STAT1 to DNA, define distinct novel genetic causes of Mendelian susceptibility to mycobacterial disease and provide further insight into the molecular mechanism of disease.

Transgenic plants expressing foreign genes are suitable systems for the production of relevant immunogens in high amounts that can be used to develop a new generation of vaccines against a variety of infectious diseases. Transgenic tobacco and potato plants expressing the nucleocapsid protein of Hantavirus serotype Puumala were generated and established. Puumala virus is a human pathogen causing hemorrhagic fever with renal syndrome. To investigate oral immunogenicity of the nucleocapsid protein expressed in plants, mice were fed with tubers of transgenic potato and tobacco leaf powder. The resulting antibodies were compared among groups. No significant difference could be found between the control group and the groups of animals, which had been fed with the recombinant plants expressing Puumala nucleocapsid protein. Hence, the effect of different enzymes, present in the gastro-intestinal tract, on the plant-derived antigen was investigated. It was found that the recombinant viral protein was completely degraded by trypsin and/or pepsin. In conclusion, the enzymes present in the intestine can degrade major antigenic domains of antigens, expressed in transgenic plants, thus preventing the induction of antibodies against the ingested viral antigen.

Transgenic plants expressing a foreign gene are a suitable system for the production of relevant immunogens in high amounts that can be used for the development of a new generation of vaccines against a variety of infectious diseases. In the present study, the expression of the nucleocapsid (N) protein of hantavirus serotype Puumala in tobacco and potato plants was investigated. Transgenic tobacco and potato plants were generated and established. These transgenic plants expressed the N protein of Puumala virus strain CG-1820. No major differences were observed when the phenotype and growth rates of transgenic plants were compared to those of normal plants. However, it was found that the leaves of transgenic tobacco plants were more slender and the tubers of transgenic potato plants were smaller than those in normal plants. In order to investigate the distribution of the expression of the foreign gene in transgenic plants, the proteins of leaves and roots of the individual transgenic tobacco and potato plants were examined by Western blot analyses. It was found that all transgenic tobacco and potato plants expressed the N protein in the leaves, whereas transgenic potato plants are able to significantly express the viral proteins also in the tubers and roots. The antigens were expressed at a level of 1 ng of protein/5 μg of dried leaves. The hantaviral recombinant N proteins obtained from transgenic tobacco and potato plants were able to elicit specific humoral and mucosal immune responses when administered intraperitoneally or orally to rabbits and mice. The expression of viral proteins in plants has two major advantages compared to other expression systems: firstly, there is no risk of contamination with mammalian viruses or other pathogens, and secondly, the production of high amounts of antigens is cheap and therefore of great economic interest. Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

TNF-receptor-associated periodic syndrome (TRAPS) is a recently recognized disorder characterized by prolonged attacks of high fever and severe localized inflammation. TRAPS is caused by dominant mutations in the 55 kDa TNF receptor gene (TNFRSF1A). We here describe three German TRAPS patients of two families with Cys30→Arg and Thr50→Met mutations, respectively. Both mutations have already been observed before in other nonrelated families. The Thr50→Met amino acid exchange, caused by an ACG→ATG transition, has been reported in two other families of different ethnic background. The possibility that the ACG→ATG sequence alteration is a mutational hot spot causing TRAPS is discussed. Furthermore, we describe and discuss the symptoms of our patients, possible inducers of febrile attacks, and treatments which the patients had received when their diagnoses were still unknown.

Chronic granulomatous disease (CGD) is an inherited disorder characterized by the inability of phagocytes to generate normal amounts of superoxide (O2 –), leaving patients susceptible to life-threatening infections. It was previously assumed that once carriers of the X-linked form of CGD were found to have 30% or more of functionally normal neutrophils, they would be free of risk for infection because the lyonization ratio was believed to be constant. Our report strongly contradicts this assumption. A 45-year-old X-CGD carrier had approximately 40% of normal neutrophils in her peripheral blood at age 21 years. Recently, she contracted a life-threatening pulmonary infection with Aspergillus fumigatus. After recovery, the ratio of normal-to-nonfunctional neutrophils was re-evaluated. She was found to have only 6–8% of normal neutrophils, suggesting that a striking decrease in the number of normal cells over the past 25 years was the reason for an increased susceptibility to Aspergillus infection. We conclude that age-related acquired skewing of the lyonization ratio can result in an increased susceptibility to life-threatening infections in X-CGD carriers.

Chronic granulomatous disease is an inherited disease characterized by the inability of phagocytes to generate normal amounts of superoxide, leaving patients susceptible to opportunistic, life-threatening infections. In the majority of cases, cytochrome b 558 is absent in the X-chromosomal form of CGD. However, the neutrophils from six of nine X-linked CGD patients, reported here, expressed normal or decreased amounts of this cytochrome and are referred to as "variant" forms. In three of these six variant patients, a roughly proportional decrease in cytochrome b 558 expression and production of H 2 O 2 were found. In two cases this phenotype could be well explained by special splice mutations, whereas in the third case it was caused by a missense mutation, predicting Ser 193 → Phe. In the other three variant patients, cytochrome b 558 expression and H 2 O 2 production were clearly disproportionate as the generation of H 2 O 2 was much more decreased than cytochrome expression. Missense mutations also were found in these cases. One of these mutations, predicting Leu 546 → Pro and affecting the putative nicotinamide adenine dinucleotide phosphate binding site, led to normal levels of cytochrome b 558 expression and reduced H 2 O 2 production. In the other two mutations, predicting Pro 339 → His and His 338 → Tyr, the putative flavin adenine dinucleotide binding site was affected. This could explain the corresponding uncommon phenotypes, characterized by zero or trace amounts of H 2 O 2 production and the expression of relatively high amounts of nonfunctional or low functional cytochrome b 558 , respectively. The only missense mutation found that prevented the expression of any cytochrome b 558 was caused by a predicted His 222 → Arg exchange in one of the three classic cases. The two other classic phenotypes were caused by splice mutations.

Patients with unexplained recurrent febrile episodes and CASP1 variants suffer from systemic sterile inflammation despite reduced secretion of IL-1s. As previously demonstrated by our group CASP1 variants lead to reduced enzymatic activity of procaspase-1 by destabilizing the tertiary structure of the caspase-1 tetramer. A possible explanation for an alternative pro-inflammatory pathway has been provided by Lamkanfi and colleagues indicating an association between enzymatically inactive procaspase-1 and receptor interacting protein kinase 2 (RIP2) leading to NF-kB activation.