UPF1 RNA Immunoprecipitation from Mini-μ Construct–expressing Cells

UPF1, an RNA helicase and a core factor of nonsense-mediated mRNA decay (NMD), interacts with RNA independently of the sequence context. To investigate the influence of translation on the association of UPF1 with specific reporter transcripts, UPF1 RNA immunoprecipitations (RIPs) are performed from Hela cells that either express a normally translated immunoglobulin-µ (Ig-µ) reporter (mini µ) or a version with a stable stem loop in the 5' UTR (SL mini µ) that efficiently inhibit translation initiation (Zund et al., 2013). Both the cloning of the SL mini µ reporter construct and the UPF1 RIP experiment are described in detail.

Two Loci on Chromosome 5 Are Associated with Serum IgE Levels in Labrador Retrievers

Crosslinking of immunoglobulin E antibodies (IgE) bound at the surface of mast cells and subsequent mediator release is considered the most important trigger for allergic reactions. Therefore, the genetic control of IgE levels is studied in the context of allergic diseases, such as asthma, atopic rhinitis, or atopic dermatitis (AD). We performed genome-wide association studies in 161 Labrador Retrievers with regard to total and allergen-specific immunoglobulin E (IgE) levels. We identified a genome-wide significant association on CFA 5 with the antigen-specific IgE responsiveness to Acarus siro. We detected a second genome-wide significant association with respect to the antigen-specific IgE responsiveness to Tyrophagus putrescentiae at a different locus on chromosome 5. A. siro and T. putrescentiae both belong to the family Acaridae and represent so-called storage or forage mites. These forage mites are discussed as major allergen sources in canine AD. No obvious candidate gene for the regulation of IgE levels is located under the two association signals. Therefore our studies offer a chance of identifying a novel mechanism controlling the host's IgE response.

Systemic light-chain amyloidosis revealed by progressive nail involvement, diffuse alopecia and sicca syndrome: report of an unusual case with a review of the literature.

Immunoglobulin light-chain (AL) amyloidosis is a form of systemic amyloidosis in which the fibrils are derived from monoclonal light chains. We report a case of a 66-year-old woman presenting with nail changes, parchment-like hand changes, progressive alopecia and sicca syndrome. Histopathological studies of biopsy specimens of the scalp, the nail, minor labial salivary glands and abdominal skin revealed deposits of AL κ-type amyloid. Urine protein electrophoresis exhibited a weak band of κ-type light chains. Based on this striking case, we here review the characteristic nail and hair manifestations associated with systemic amyloidosis. Knowledge of these signs is important for an early diagnosis of systemic amyloidosis, identification of the underlying disease and patient management.

Different IVIG glycoforms affect in vitro inhibition of anti-ganglioside antibody-mediated complement deposition

Intravenous immunoglobulin (IVIG) is the first line treatment for Guillain–Barré syndrome and multifocal motor neuropathy, which are caused by anti-ganglioside antibody-mediated complement-dependent cytotoxicity. IVIG has many potential mechanisms of action, and sialylation of the IgG Fc portion reportedly has an anti-inflammatory effect in antibody-dependent cell-mediated cytotoxicity models. We investigated the effects of different IVIG glycoforms on the inhibition of antibody-mediated complement-dependent cytotoxicity. Deglycosylated, degalactosylated, galactosylated and sialylated IgG were prepared from IVIG following treatment with glycosidases and glycosyltransferases. Sera from patients with Guillain–Barré syndrome, Miller Fisher syndrome and multifocal motor neuropathy associated with anti-ganglioside antibodies were used. Inhibition of complement deposition subsequent to IgG or IgM autoantibody binding to ganglioside, GM1 or GQ1b was assessed on microtiter plates. Sialylated and galactosylated IVIGs more effectively inhibited C3 deposition than original IVIG or enzyme-treated IVIGs (agalactosylated and deglycosylated IVIGs). Therefore, sialylated and galactosylated IVIGs may be more effective than conventional IVIG in the treatment of complement-dependent autoimmune diseases.

A glance on recent progresses in diagnosis and treatment of primary immunodeficiencies

Primary immunodeficiencies (PIDs)* belong to the group of rare diseases which need more awareness by the relevant medical disciplines. Below a review on recent progresses in diagnosis and treatment of PIDs is given. Reducing the regrettable delay in diagnosis of PIDs (worldwide) is possible only when awareness is increased by doctors who may encounter patients with PID. This review shall serve this purpose. Progresses in understanding what the link might be between one genetic defect presenting in various phenotypes or how various gene defects may manifest by very similar PID phenotypes helps building awareness. Knowledge of PID favours early diagnosis, a cornerstone of optimal, sometimes life-long care at justifiable costs. The complexity of PIDs calls for clinical laboratory and clinical diagnostic performed by experts only. Exciting laboratory diagnostic progresses in early diagnosis of the most severe forms of PID are reviewed below. Progresses in curative therapies for PIDs, such as hematopoietic stem cell transplantation and gene therapies, are mentioned in short. About 80% of PID patients suffer from an antibody deficiency syndrome and can profit from non-curative replacement therapies with human immunoglobulin G concentrates. Modes of application, safety and hints for dosing of replacement therapies to reduce frequencies of severe infections are mentioned below. Thanks to the increasing quality of care, patients survive adolescence. A glance is given on the problems of transition to the adult medicine setting.

Use of dextran sulfate in tourniquet-induced skeletal muscle reperfusion injury.

BACKGROUND Lower extremity ischemia-reperfusion injury (IRI)-prolonged ischemia and the subsequent restoration of circulation-may result from thrombotic occlusion, embolism, trauma, or tourniquet application in surgery. The aim of this study was to assess the effect of low-molecular-weight dextran sulfate (DXS) on skeletal muscle IRI. METHODS Rats were subjected to 3 h of ischemia and 2 or 24 h of reperfusion. To induce ischemia the femoral artery was clamped and a tourniquet placed under the maintenance of the venous return. DXS was injected systemically 10 min before reperfusion. Muscle and lung tissue samples were analyzed for deposition of immunoglobulin M (IgM), IgG, C1q, C3b/c, fibrin, and expression of vascular endothelial-cadherin and bradykinin receptors b1 and b2. RESULTS Antibody deposition in reperfused legs was reduced by DXS after 2 h (P < 0.001, IgM and IgG) and 24 h (P < 0.001, IgM), C3b/c deposition was reduced in muscle and lung tissue (P < 0.001), whereas C1q deposition was reduced only in muscle (P < 0.05). DXS reduced fibrin deposits in contralateral legs after 24 h of reperfusion but did not reduce edema in muscle and lung tissue or improve muscle viability. Bradykinin receptor b1 and vascular endothelial-cadherin expression were increased in lung tissue after 24 h of reperfusion in DXS-treated and non-treated rats but bradykinin receptor b2 was not affected by IRI. CONCLUSIONS In contrast to studies in myocardial infarction, DXS did not reduce IRI in this model. Neither edema formation nor viability was improved, whereas deposition of complement and coagulation components was significantly reduced. Our data suggest that skeletal muscle IRI may not be caused by the complement or coagulation alone, but the kinin system may play an important role.

Refinement of tourniquet-induced peripheral ischemia/reperfusion injury in rats: comparison of 2 h vs 24 h reperfusion.

Prolonged ischemia of skeletal muscle tissue, followed by reperfusion, leads to ischemia/reperfusion injury (IRI), which is a feared local and systemic inflammatory reaction. With respect to the 3Rs, we wanted to determine which parameters for assessment of IRI require a reperfusion time of 24 h and for which 2 h of reperfusion are sufficient. Rats were subjected to 3 h of hind limb ischemia and 2 h or 24 h of reperfusion. Human plasma derived C1 inhibitor was used as a drug to prevent reperfusion injury. For 2 h of reperfusion the rats stayed under anesthesia throughout (severity grade 1), whereas for 24 h they were awake under analgesia during reperfusion (grade 2). The femoral artery was clamped and a tourniquet was placed, under maintenance of venous return. C1 esterase inhibitor was systemically administered 5 min before the induction of ischemia. No differences in local muscle edema formation and depositions of immunoglobulin G and immunoglobulin M were observed between 2 h and 24 h (P > 0.05), whereas lung edema was only observed after 24 h. Muscle viability was significantly lower after 24 h vs 2 h reperfusion (P < 0.05). Increased plasma creatine kinase (CK)-MM and platelet-derived growth factor (PDGF)-bb could be detected after 2 h, but not after 24 h of reperfusion. By contrast, depositions of C3b/c and fibrin in muscle were only detected after 24 h (P < 0.001). In conclusion, for a first screening of drug candidates to reduce IRI, 2 h reperfusions are sufficient, and these reduce the severity of the animal experiment. Twenty-four-hour reperfusions are only needed for in-depth analysis of the mechanisms of IRI, including lung damage.

The splenic autoimmune response to ADAMTS13 in thrombotic thrombocytopenic purpura contains recurrent antigen-binding CDR3 motifs.

Acquired thrombotic thrombocytopenic purpura (TTP) is the consequence of a severe ADAMTS13 deficiency resulting from autoantibodies inhibiting ADAMTS13 or accelerating its clearance. Despite the success of plasma exchange the risk of relapse is high. From 2 patients (A and B), splenectomized for recurrent episodes of acquired TTP, the splenic B-cell response against ADAMTS13 was characterized through generation of human monoclonal anti-ADAMTS13 autoantibodies (mAbs) by cloning an immunoglobulin G (IgG)4κ- and IgG4λ-Fab library using phage display technology and by Epstein-Barr virus transformation of switched memory B cells (CD19+/CD27+/IgG+). Sequence analysis of the anti-ADAMTS13 IgGs of both patients revealed that the VH gene use was limited in our patients to VH1-3 (55%), VH1-69 (17%), VH3-30 (7%), and VH4-28 (21%) and contained 8 unique and thus far not reported heavy-chain complementarity determining region 3 motifs, of which 4 were shared by the 2 patients. The discovery of several highly similar anti-ADAMTS13 autoantibodies in 2 unrelated TTP patients suggests that the autoimmune response is antigen driven, because the probability that such similar immunoglobulin rearrangements happen by chance is very low (< 10(-9)).

Colostrogenesis: IgG1 Transcytosis Mechanisms

Biological transport of intact proteins across epithelial cells has been documented for many absorptive and secretory tissues. Immunoglobulins were some of the earliest studied proteins in this category. The transcellular transport (transcytosis) of immunoglobulins in neonatal health and development has been recognized; the process is especially significant with ungulates because they do not transcytose immunoglobulins across the placenta to the neonate. Rather, they depend upon mammary secretion of colostrum and intestinal absorption of immunoglobulins in order to provide intestinal and systemic defense until the young ungulate develops its own humoral defense mechanisms. The neonatal dairy calf's ability to absorb immunoglobulins from colostrum is assisted by a ~24 h "open gut" phenomenon where large proteins pass the intestinal epithelial cells and enter the systemic system. However, a critical problem recognized for newborn dairy calves is that an optimum mass of colostrum Immunoglobulin G (IgG) needs to be absorbed within that 24 h window in order to provide maximal resistance to disease. Many calves do not achieve the optimum because of poor quality colostrum. While many studies have focused on calf absorption, the principal cause of the problem resides with the extreme variation (g to kg) in the mammary gland's capacity to transfer blood IgG1 into colostrum. Colostrum is a unique mammary secretory product that is formed during late pregnancy when mammary cells are proliferating and differentiating in preparation for lactation. In addition to the transcytosis of immunoglobulins, the mammary gland also concentrates a number of circulating hormones into colostrum. Remarkably, the mechanisms in the formation of colostrum in ungulates have been rather modestly studied. The mechanisms and causes of this variation in mammary gland transcytosis of IgG1 are examined, evaluated, and in some cases, explained

Colour measurement of colostrum for estimation of colostral IgG and colostrum composition in dairy cows

Instruments for on-farm determination of colostrum quality such as refractometers and densimeters are increasingly used in dairy farms. The colour of colostrum is also supposed to reflect its quality. A paler or mature milk-like colour is associated with a lower colostrum value in terms of its general composition compared with a more yellowish and darker colour. The objective of this study was to investigate the relationships between colour measurement of colostrum using the CIELAB colour space (CIE L*=from white to black, a*=from red to green, b*=from yellow to blue, chroma value G=visual perceived colourfulness) and its composition. Dairy cow colostrum samples (n=117) obtained at 4·7±1·5 h after parturition were analysed for immunoglobulin G (IgG) by ELISA and for fat, protein and lactose by infrared spectroscopy. For colour measurements, a calibrated spectrophotometer was used. At a cut-off value of 50 mg IgG/ml, colour measurement had a sensitivity of 50·0%, a specificity of 49·5%, and a negative predictive value of 87·9%. Colostral IgG concentration was not correlated with the chroma value G, but with relative lightness L*. While milk fat content showed a relationship to the parameters L*, a*, b* and G from the colour measurement, milk protein content was not correlated with a*, but with L*, b*, and G. Lactose concentration in colostrum showed only a relationship with b* and G. In conclusion, parameters of the colour measurement showed clear relationships to colostral IgG, fat, protein and lactose concentration in dairy cows. Implementation of colour measuring devices in automatic milking systems and milking parlours might be a potential instrument to access colostrum quality as well as detecting abnormal milk.

Continuous milking of dairy cows disrupts timing of peak IgG concentration appearance in mammary secretions

The length of the dry period in commercial dairy production is under close scrutiny. While the main concern is the composition and volume of milk produced, the evaluation of colostrum quality under these new paradigms has suggested a decline in IgG concentrations, while some reports indicate no change. Colostrum quality has been defined as an adequate concentration (>50 mg/ml) of immunoglobulin in the secretions to provide the newborn with maximal disease resistance. We investigated the appearance of IgG in mammary pre- and post partum secretions in cows without a dry period (continuously milked, Dry0) and compared the secretions with cows that experienced a dry period of 60 d (Dry60). Blood was collected during the experimental period and plasma analysed for progesterone (P4) and prolactin (Prl). Approximately -6 d relative to parturition, the Dry0 animals exhibited increased concentration of IgG in their secretions to an average of ∼35 mg/ml that remained rather constant through subsequent pregnancy and following parturition. Dry0 cows were producing an average IgG concentration in parturition colostrum of 44·2±17·6 mg/ml that was not different than that of controls (66·86±16·8 mg/ml). However, Dry0 cows exhibited high variation, different peak times (day) of IgG concentration including times that occurred both pre and post parturition. IgG mass of the Dry0 cows remained rather constant pre- and post partum and did not show the same declining mass following parturition that was shown for the Dry60 cows. The change in plasma P4 and Prl were shown to have no timing effect on colostrum IgG concentration.

New findings on echinococcosis

The following new aspects of cystic and alveolar echinococcosis (= infections with the metacestode stages of Echinococcus granulosus and E. multilocularis respectively) are reported: identification of a Swiss E. granulosus isolate as "cattle strain" which differs from the "sheep strain"; new observations on proliferation and metastasis formation of larval E. multilocularis; information on chemotherapy of human echinococcosis; recent developments in immunoserology. The latter includes a new technique for serological differential diagnosis of cystic and alveolar echinococcosis, the determination of parasite-specific immunoglobulin classes (IgG, IgM, IgA and IgE) and circulating antigens in ELISA, and the introduction of arc-5 detection in routine serodiagnosis. A highly purified, species-specific antigen from E. multilocularis is now available for seroepidemiological studies.

Segmented filamentous bacterium uses secondary and tertiary lymphoid tissues to induce gut IgA and specific T helper 17 cell responses.

Segmented filamentous bacterium (SFB) is a symbiont that drives postnatal maturation of gut adaptive immune responses. In contrast to nonpathogenic E. coli, SFB stimulated vigorous development of Peyer's patches germinal centers but paradoxically induced only a low frequency of specific immunoglobulin A (IgA)-secreting cells with delayed accumulation of somatic mutations. Moreover, blocking Peyer's patch development abolished IgA responses to E. coli, but not to SFB. Indeed, SFB stimulated the postnatal development of isolated lymphoid follicles and tertiary lymphoid tissue, which substituted for Peyer's patches as inductive sites for intestinal IgA and SFB-specific T helper 17 (Th17) cell responses. Strikingly, in mice depleted of gut organized lymphoid tissue, SFB still induced a substantial but nonspecific intestinal Th17 cell response. These results demonstrate that SFB has the remarkable capacity to induce and stimulate multiple types of intestinal lymphoid tissues that cooperate to generate potent IgA and Th17 cell responses displaying only limited target specificity.

Inflammatory, immunological, and intestinal disease biomarkers in Chinese Shar-Pei dogs with marked hypocobalaminemia.

Chinese Shar-Pei dogs have a high prevalence of hypocobalaminemia and are commonly presented with clinical signs suggestive of severe and long-standing gastrointestinal disease such as diarrhea, vomiting, and/or weight loss. The aim of the current study was to evaluate serum concentrations of inflammatory markers, markers for intestinal disease, and immunological markers in Shar-Peis with hypocobalaminemia or normocobalaminemia (serum cobalamin concentrations within the reference interval). Serum samples from Shar-Peis were collected from various parts of the United States. Serum concentrations of inflammatory markers (i.e., C-reactive protein [CRP], calprotectin [CP], and S100A12), hyaluronic acid (HA, a marker for cutaneous mucinosis), and analytes commonly altered in chronic intestinal diseases (i.e., albumin, zinc, alpha1-proteinease inhibitor [α1PI], immunoglobulin [Ig]A, and IgM) were compared between Shar-Peis with hypocobalaminemia and Shar-Peis with normocobalaminemia. Serum concentrations of CRP, CP, S100A12, HA, zinc, and cα1-PI concentrations did not differ between hypocobalaminemic and normocobalaminemic Shar-Peis (P > 0.05). Serum concentrations of albumin were significantly lower in hypocobalaminemic Shar-Peis (median: 2.5 g/dl) than in normocobalaminemic Shar-Peis (median: 2.9 g/dl; P < 0.0001). Higher serum IgA concentrations and lower serum IgM concentrations were observed in hypocobalaminemic Shar-Peis (median: 1.7 g/l and 0.8 g/l, respectively) than in normocobalaminemic Shar-Peis (median: 0.7 g/l and 1.9 g/l, respectively; both P < 0.0001). In conclusion, no difference was found in serum concentrations of CRP, CP, S100A12, and HA between hypocobalaminemic and normocobalaminemic Shar-Peis whereas some differences were observed in analytes (e.g., albumin, IgA, and IgM) that may be altered in patients with chronic enteropathies.

Transgenic Expression of Human CD46 on Porcine Endothelium: Effect on Coagulation and Fibrinolytic Cascades During Ex Vivo Human-to-Pig Limb Xenoperfusions

BACKGROUND Dysregulation of the coagulation system due to inflammatory responses and cross-species molecular incompatibilities represents a major obstacle to successful xenotransplantation. We hypothesized that complement inhibition mediated by transgenic expression of human CD46 in pigs might also regulate the coagulation and fibrinolysis cascades and tested this in ex vivo human-to-pig xenoperfusions. METHODS Forelimbs of wild-type and hCD46/HLA-E double transgenic pigs were ex vivo xenoperfused for 12 hours with whole heparinized human blood. Muscle biopsies were stained for galactose-α1,3-galactose, immunoglobulin M, immunoglobulin G, complement, fibrin, tissue factor, fibrinogen-like protein 2, tissue plasminogen activator (tPA), and plasminogen activator inhibitor (PAI)-1. The PAI-1/tPA complexes, D-dimers, and prothrombin fragment F1 + 2 were measured in plasma samples after ex vivo xenoperfusion. RESULTS No differences of galactose expression or deposition of immunoglobulin M and immunoglobulin G were found in xenoperfused tissues of wild type and transgenic limbs. In contrast, significantly lower deposition of C5b-9 (P < 0.0001), fibrin (P = 0.009), and diminished expression of tissue factor (P = 0.005) and fibrinogen-like protein 2 (P = 0.028) were found in xenoperfused tissues of transgenic limbs. Levels of prothrombin fragment F1 + 2 (P = 0.031) and D-dimers (P = 0.044) were significantly lower in plasma samples obtained from transgenic as compared to wild-type pig limb perfusions. The expression of the fibrinolytic marker tPA was significantly higher (P = 0.009), whereas PAI-1 expression (P = 0.022) and PAI-1/tPA complexes in plasma (P = 0.015) were lower after transgenic xenoperfusion as compared to wild-type xenoperfusions. CONCLUSIONS In this human-to-pig xenoperfusion model, complement inhibition by transgenic hCD46 expression led to a significant inhibition of procoagulant and antifibrinolytic pathways.