Evidence that High-Affinity IgE Can Develop in the Germinal Center in the Absence of an IgG1-Switched Intermediate.

High-affinity allergen-specific IgE is essential for the severe allergic anaphylaxis response. High-affinity Abs are formed by successive rounds of selection of Ag-specific B cells in the germinal center (GC); however, several studies have shown that IgE+ GC B cells are impaired in their ability to undergo selection in the GC. A pathway, known as the "indirect switching pathway" for IgE, has been described whereby Ag-specific B cells initially switch to the IgG1 isotype and undergo affinity selection in the GC, with a secondary switch to the IgE isotype after affinity selection. In previous work, using a food allergy model in mice, we investigated how high-affinity IgE develops in the GC, but we did not test the indirect switching model. In this study, we analyzed the importance of the indirect switching pathway by constructing IgG1-cre Bcl6-fl/fl mice. In these mice, once B cells switch to IgG1, they delete Bcl6 and thus cannot enter or persist in the GC. When we tested IgG1-cre Bcl6-fl/fl mice with our food allergy model, we found that, as expected, IgG1 Abs had decreased affinity, but unexpectedly, the affinity of IgE for allergen was unchanged. IgG1-cre Bcl6-fl/fl mice underwent anaphylaxis in response to allergen, consistent with the formation of high-affinity IgE. Thus, in a food allergy response, high-affinity IgE can be efficiently formed in the absence of indirect switching to IgG1, either by direct selection of IgE+ GC B cells or indirect selection of IgM+ GC B cells that later switch to IgE.

Reliability and validity of the German translation of the de Morton Mobility Index (DEMMI) performed by physiotherapists in patients admitted to a sub-acute inpatient geriatric rehabilitation hospital.

BACKGROUND: Mobility is a key outcome in geriatric rehabilitation. The de Morton Mobility Index (DEMMI) is an internationally well-established, unidimensional measure of mobility with good psychometric properties. The aim of this study was to examine the reliability and construct validity of the German translation of the DEMMI in geriatric inpatients. METHODS: This cross-sectional study included patients admitted to a sub-acute inpatient geriatric rehabilitation hospital (reliability sample: N = 33; validity sample: N = 107). Reliability, validity, and unidimensionality were investigated. RESULTS: Inter-rater reliability between two graduate physiotherapists was excellent, with intra-class correlation coefficient of 0.94 (95% confidence interval: 0.88-0.97). The minimal detectable change with 90% confidence was 9 points. Construct validity for the DEMMI was evidenced by significant moderate to strong correlations with other measures of mobility and related constructs (Performance Oriented Mobility Assessment: rho = 0.89; Functional Ambulation Categories: rho = 0.70; six-minute walk test: rho = 0.73; gait speed: rho = 0.67; Falls Efficacy Scale International: rho = -0.68). Known-groups validity was indicated by significant DEMMI mean group differences between independent versus dependent walkers and walking aid users versus non-users. Unidimensionality of the German DEMMI translation was confirmed by Rasch analysis. CONCLUSIONS: The German translation of the DEMMI is a unidimensional instrument producing valid and reproducible measurement of mobility in an inpatient geriatric rehabilitation setting.

Role of human Kallistatin in glucose and energy homeostasis in mice.

OBJECTIVE: Kallistatin (KST), also known as SERPIN A4, is a circulating, broadly acting human plasma protein with pleiotropic properties. Clinical studies in humans revealed reduced KST levels in obesity. The exact role of KST in glucose and energy homeostasis in the setting of insulin resistance and type 2 diabetes is currently unknown. METHODS: Kallistatin mRNA expression in human subcutaneous white adipose tissue (sWAT) of 47 people with overweight to obesity of the clinical trial "Comparison of Low Fat and Low Carbohydrate Diets With Respect to Weight Loss and Metabolic Effects (B-SMART)" was measured. Moreover, we studied transgenic mice systemically overexpressing human KST (hKST-TG) and wild type littermate control mice (WT) under normal chow (NCD) and high-fat diet (HFD) conditions. RESULTS: In sWAT of people with overweight to obesity, KST mRNA increased after diet-induced weight loss. On NCD, we did not observe differences between hKST-TG and WT mice. Under HFD conditions, body weight, body fat and liver fat content did not differ between genotypes. Yet, during intraperitoneal glucose tolerance tests (ipGTT) insulin excursions and HOMA-IR were lower in hKST-TG (4.42 ± 0.87 AU, WT vs. 2.20 ± 0.27 AU, hKST-TG, p < 0.05). Hyperinsulinemic euglycemic clamp studies with tracer-labeled glucose infusion confirmed improved insulin sensitivity by higher glucose infusion rates in hKST-TG mice (31.5 ± 1.78 mg/kg/min, hKST-TG vs. 18.1 ± 1.67 mg/kg/min, WT, p < 0.05). Improved insulin sensitivity was driven by reduced hepatic insulin resistance (clamp hepatic glucose output: 7.7 ± 1.9 mg/kg/min, hKST-TG vs 12.2 ± 0.8 mg/kg/min, WT, p < 0.05), providing evidence for direct insulin sensitizing effects of KST for the first time. Insulin sensitivity was differentially affected in skeletal muscle and adipose tissue. Mechanistically, we observed reduced Wnt signaling in the liver but not in skeletal muscle, which may explain the effect. CONCLUSIONS: KST expression increases after weight loss in sWAT from people with obesity. Furthermore, human KST ameliorates diet-induced hepatic insulin resistance in mice, while differentially affecting skeletal muscle and adipose tissue insulin sensitivity. Thus, KST may be an interesting, yet challenging, therapeutic target for patients with obesity and insulin resistance.

Genetically programmed alternative splicing of NEMO mediates an autoinflammatory disease phenotype.

Host defense and inflammation are regulated by the NF-κB essential modulator (NEMO), a scaffolding protein with a broad immune cell and tissue expression profile. Hypomorphic mutations in inhibitor of NF-κB kinase regulatory subunit gamma (IKBKG) encoding NEMO typically present with immunodeficiency. Here, we characterized a pediatric autoinflammatory syndrome in 3 unrelated male patients with distinct X-linked IKBKG germline mutations that led to overexpression of a NEMO protein isoform lacking the domain encoded by exon 5 (NEMO-Δex5). This isoform failed to associate with TANK binding kinase 1 (TBK1), and dermal fibroblasts from affected patients activated NF-κB in response to TNF but not TLR3 or RIG-I-like receptor (RLR) stimulation when isoform levels were high. By contrast, T cells, monocytes, and macrophages that expressed NEMO-Δex5 exhibited increased NF-κB activation and IFN production, and blood cells from these patients expressed a strong IFN and NF-κB transcriptional signature. Immune cells and TNF-stimulated dermal fibroblasts upregulated the inducible IKK protein (IKKi) that was stabilized by NEMO-Δex5, promoting type I IFN induction and antiviral responses. These data revealed how IKBKG mutations that lead to alternative splicing of skipping exon 5 cause a clinical phenotype we have named NEMO deleted exon 5 autoinflammatory syndrome (NDAS), distinct from the immune deficiency syndrome resulting from loss-of-function IKBKG mutations.

[Assessment of the significance and the requirements of medical postdoctoral lecture qualifications in Germany by the assessment committees]. / Bewertung des Stellenwerts und der Voraussetzungen der medizinischen Habilitation in Deutschland durch die beurteilenden Kommissionen.

OBJECTIVE: The postdoctoral medical lecture qualification (Habilitation) represents the highest academic qualification in Germany, which is successfully completed by approximately 850 candidates in medicine and health sciences per year. However, there is only a limited number of respective academic positions available. In addition, structures in education and society have changed over the last years, challenging the importance of this specific German qualification. The aim of this study was to elicit the opinions of members of German habilitation committees concerning the requirements, processes and the overall importance of the postdoctoral medical lecture qualification. Furthermore we wanted to evaluate potential needs for reforms. METHODS: The online survey was conducted asking for biographic parameters, subjective ratings and potential needs for reforms concerning the postdoctoral medical lecture qualification (PLQ). RESULTS: The PLQ was rated high in significance by 71.3 % of the committee members. According to the medical understanding of the Humboldt triad (research, teaching, patient care), research (94.3 %) and teaching (89.7 %) have been rated as the most important requirements for a PLQ. Asked for the motivation to undertake a PLQ, 91.0 % of the members gave the joy of doing research, 78.2 % the joy of teaching and 65.5 % better career prospects perspectives as their reason. The recognition of a Ph.D. degree as being equivalent to a German PQL was clearly rejected by the survey respondents (58.6 %: no equivalence). The majority is against the abolition of the German PLQ. However, there is a definite desire for reform, preferably concerning internal obstacles such as the dependence on full professors, more transparency in the PLQ process, but also the demand for a federal standard PLQ regulation. CONCLUSION: From the committee members' point of view the German PLQ has still a role to play and, despite controversy, is still regarded as a timely qualification. However, there is clear-cut evidence for local, national and international reforms in order to create equality of opportunity for the candidates and to open up suitable career options.

Host susceptibility to gram-negative pneumonia after lung contusion.

BACKGROUND: Lung contusion (LC) induces inflammation with high local concentrations of proinflammatory mediators stimulating chemotaxis and activation of neutrophils. LC is also a risk factor for development of pneumonia; however, the reason for this increased susceptibility is not clearly identified. We hypothesize that LC creates acute changes in the host pulmonary innate immune system that leads to vulnerability from a "second" hit bacterial infection. METHODS: Female C57Bl/6 mice underwent LC injury at time -6 hours. At 0 hours, these mice were inoculated intratracheally with 1,000 colony forming unit (CFU) of Klebsiella pneumoniae (LC+Pneu) or vehicle (LC). Control animals underwent a sham LC injury followed by pneumonia (Sham+Pneu). Bronchoalveolar lavage (BAL) fluid and lung tissue specimens were collected. Lung bacteria levels were quantified by serial dilution, plating, and counting CFUs. Cytokine levels were assayed by ELISA. Cell type identification and quantification was performed using flow cytometry. RESULTS: Survival at 72 hours was markedly different for the LC, Sham+Pneu, and LC+Pneu groups (100%, 80%, 20%, p < 0.05 Sham+Pneu vs. LC+Pneu). LC+Pneu animals had decreased pulmonary bacterial clearance at 24 hours compared with the Sham+Pneu group (4 × 10(7) vs. 8 × 10(6) CFUs, p < 0.05). BAL levels of IL-1ß, IL-6, and keratinocyte chemoattractant were all significantly elevated in LC+Pneu mice compared with the Sham+Pneu group at 24 hours. Conversely, the Sham+Pneu mice had increased levels of macrophage inflammatory protein-2, total cells, macrophages, and neutrophils in BAL compared with the LC+Pneu group at 24 hours. LC+Pneu animals demonstrated changes in macrophage apoptosis and necrosis in BAL samples obtained 2 hours after induction of pneumonia when compared with the Sham+Pneu group. Both Sham+Pneu and LC+Pneu animals demonstrated an increase in the level of IL-10 in BAL fluid compared with LC animals. CONCLUSION: Acute inflammation after LC acts to modulate the presence of inflammatory cells necessary to combat gram-negative bacteria. This results in decreased bacterial clearance and increased mortality from pneumonia.

Natriuretic peptides enhance the oxidative capacity of human skeletal muscle.

Cardiac natriuretic peptides (NP) are major activators of human fat cell lipolysis and have recently been shown to control brown fat thermogenesis. Here, we investigated the physiological role of NP on the oxidative metabolism of human skeletal muscle. NP receptor type A (NPRA) gene expression was positively correlated to mRNA levels of PPARγ coactivator-1α (PGC1A) and several oxidative phosphorylation (OXPHOS) genes in human skeletal muscle. Further, the expression of NPRA, PGC1A, and OXPHOS genes was coordinately upregulated in response to aerobic exercise training in human skeletal muscle. In human myotubes, NP induced PGC-1α and mitochondrial OXPHOS gene expression in a cyclic GMP-dependent manner. NP treatment increased OXPHOS protein expression, fat oxidation, and maximal respiration independent of substantial changes in mitochondrial proliferation and mass. Treatment of myotubes with NP recapitulated the effect of exercise training on muscle fat oxidative capacity in vivo. Collectively, these data show that activation of NP signaling in human skeletal muscle enhances mitochondrial oxidative metabolism and fat oxidation. We propose that NP could contribute to exercise training-induced improvement in skeletal muscle fat oxidative capacity in humans.