Persistent Autoantibody-Production by Intermediates between Short-and Long-Lived Plasma Cells in Inflamed Lymph Nodes of Experimental Epidermolysis Bullosa Acquisita

Autoantibodies are believed to be maintained by either the continuous generation of short-lived plasma cells in secondary lymphoid tissues or by long-lived plasma cells localized in bone marrow and spleen. Here, we show in a mouse model for the autoimmune blistering skin disease epidermolysis bullosa acquisita (EBA) that chronic autoantibody production can also be maintained in inflamed lymph nodes, by plasma cells exhibiting intermediate lifetimes. After EBA induction by immunization with a mCOL7c-GST-fusion protein, antigen-specific plasma cells and CD4 T cells were analyzed. Plasma cells were maintained for months in stable numbers in the draining lymph nodes, but not in spleen and bone marrow. In contrast, localization of mCOL7c-GST -specific CD4 T cells was not restricted to lymph nodes, indicating that availability of T cell help does not limit plasma cell localization to this site. BrdU-incorporation studies indicated that pathogenic mCOL7c- and non-pathogenic GST-specific plasma cells resemble intermediates between short-and long-lived plasma cells with half-lives of about 7 weeks. Immunization with mCOL7c-GST also yielded considerable numbers of plasma cells neither specific for mCOL7c- nor GST. These bystander-activated plasma cells exhibited much shorter half-lives and higher population turnover, suggesting that plasma cell lifetimes were only partly determined by the lymph node environment but also by the mode of activation. These results indicate that inflamed lymph nodes can harbor pathogenic plasma cells exhibiting distinct properties and hence may resemble a so far neglected site for chronic autoantibody production.     

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.     

The Systemic Immune Network in Recent Onset Type 1 Diabetes: Central Role of Interleukin-1 Receptor Antagonist (DIATOR Trial)

Background

The hypothesis was tested that the systemic immune milieu in recent-onset type 1 diabetes is associated with residual beta cell function and other metabolic patient characteristics.

Methods and Findings

All patients (n = 89, 40% female) of the Diabetes and Atorvastatin (DIATOR) Trial were analyzed at recruitment, i.e. prior to receiving the study medication. Inclusion criteria were insulin dependent diabetes for 2 weeks to 3 months, age range 18–39 years, and islet cell autoantibodies. Blood samples were analyzed for 14 immune mediators by standard methods. Concentrations of all mediators correlated with at least one other mediator (p<0.05, Spearman correlation) giving rise to a network. Interleukin 1 receptor antagonist (IL1-RA) held a central position and was associated with both pro- and anti-inflammatory mediators. Further central elements were the pro-inflammatory mediators CRP and IL-6, the soluble adhesion molecules sICAM-1 and E-selectin, and MCP-4 which held a central position in the chemokine network. The two Th1-associated mediators IFNγ and IP-10 remained outside the network but correlated with each other. All correlations were positive (r = 0.25–0.72), i.e., high levels of pro-inflammatory mediators were accompanied by increased levels of anti-inflammatory mediators. IL-1RA was the only mediator associated with fasting and liquid mixed meal stimulated C-peptide concentrations (r = 0.31 and 0.24, p = 0.003 and 0.025, after adjustment for age, sex, BMI). There were associations between the immune mediator network and BMI (IL-1RA, CRP, IL-6, MCP-4, MIP-1ß) but few or no associations with HbA1c, insulin dose, lipid parameters, age or sex.

Conclusions

In patients with recent onset type 1 diabetes, systemic acute phase proteins, cytokines, chemokines and soluble adhesion molecules form a network. Among the few central elements IL-1RA has a dominant role. IL-1RA is associated with all other groups of mediators and is the only mediator which correlates (positively) with residual beta cell function.

Trial registration

ClinicalTrials.gov registration number: {"type":"clinical-trial","attrs":{"text":"NCT00974740","term_id":"NCT00974740"}}NCT00974740     

Veillonella,Firmicutes: Microbes disguised as Gram negatives

The Firmicutes represent a major component of the intestinal microflora. The intestinal Firmicutes are a large, diverse group of organisms, many of which are poorly characterized due to their anaerobic growth requirements. Although most Firmicutes are Gram positive, members of the class Negativicutes, including the genus Veillonella, stain Gram negative. Veillonella are among the most abundant organisms of the oral and intestinal microflora of animals and humans, in spite of being strict anaerobes. In this work, the genomes of 24 Negativicutes, including eight Veillonella spp., are compared to 20 other Firmicutes genomes; a further 101 prokaryotic genomes were included, covering 26 phyla. Thus a total of 145 prokaryotic genomes were analyzed by various methods to investigate the apparent conflict of the Veillonella Gram stain and their taxonomic position within the Firmicutes. Comparison of the genome sequences confirms that the Negativicutes are distantly related to Clostridium spp., based on 16S rRNA, complete genomic DNA sequences, and a consensus tree based on conserved proteins. The genus Veillonella is relatively homogeneous: inter-genus pair-wise comparison identifies at least 1,350 shared proteins, although less than half of these are found in any given Clostridium genome. Only 27 proteins are found conserved in all analyzed prokaryote genomes. Veillonella has distinct metabolic properties, and significant similarities to genomes of Proteobacteria are not detected, with the exception of a shared LPS biosynthesis pathway. The clade within the class Negativicutes to which the genus Veillonella belongs exhibits unique properties, most of which are in common with Gram-positives and some with Gram negatives. They are only distantly related to Clostridia, but are even less closely related to Gram-negative species. Though the Negativicutes stain Gram-negative and possess two membranes, the genome and proteome analysis presented here confirm their place within the (mainly) Gram positive phylum of the Firmicutes. Further studies are required to unveil the evolutionary history of the Veillonella and other Negativicutes.     

Repeated H2O2 exposure drives cell cycle progression in an in vitro model of ulcerative colitis

The production of hydrogen peroxide (H₂O₂) drives tumourigenesis in ulcerative colitis (UC). Recently, we showed that H₂O₂ activates DNA damage checkpoints in human colonic epithelial cells (HCEC) through c‐Jun N‐terminal Kinases (JNK) that induces p21^WAF1. Moreover, caspases circumvented the G1/S and intra‐S checkpoints, and cells accumulated in G2/M. The latter observation raised the question of whether repeated H₂O₂ exposures alter JNK activation, thereby promoting a direct passage of cells from G2/M arrest to driven cell cycle progression. Here, we report that increased proliferation of repeatedly H₂O₂‐exposed HCEC cells (C‐cell cultures) was associated with (i) increased phospho‐p46 JNK, (ii) decreased total JNK and phospho‐p54 JNK and (iii) p21^WAF1 down‐regulation. Altered JNK activation and p21^WAF1 down‐regulation were accompanied by defects in maintaining G2/M and mitotic spindle checkpoints through adaptation, as well as by apoptosis resistance following H₂O₂ exposure. This may cause increased proliferation of C‐cell cultures, a defining initiating feature in the inflammation‐carcinoma pathway in UC. We further suggest that dysregulated JNK activation is attributed to a non‐apoptotic function of caspases, causing checkpoint adaptation in C‐cell cultures. Additionally, loss of cell‐contact inhibition and the overcoming of senescence, hallmarks of cancer, contributed to increased proliferation. Furthermore, there was evidence that p54 JNK inactivation is responsible for loss of cell‐contact inhibition. We present a cellular model of UC and suggest a sinusoidal pattern of proliferation, which is triggered by H₂O₂‐induced reactive oxygen species generation, involving an interplay between JNK activation/inactivation, p21^WAF1, c‐Fos, c‐Jun/phospho‐c‐Jun, ATF2/phospho‐ATF2, β‐catenin/TCF4‐signalling, c‐Myc, CDK6 and Cyclin D2, leading to driven cell cycle progression.     

A Hereditary Spastic Paraplegia Mouse Model Supports a Role of ZFYVE26/SPASTIZIN for the Endolysosomal System

Hereditary spastic paraplegias (HSPs) are characterized by progressive weakness and spasticity of the legs because of the degeneration of cortical motoneuron axons. SPG15 is a recessively inherited HSP variant caused by mutations in the ZFYVE26 gene and is additionally characterized by cerebellar ataxia, mental decline, and progressive thinning of the corpus callosum. ZFYVE26 encodes the FYVE domain-containing protein ZFYVE26/SPASTIZIN, which has been suggested to be associated with the newly discovered adaptor protein 5 (AP5) complex. We show that Zfyve26 is broadly expressed in neurons, associates with intracellular vesicles immunopositive for the early endosomal marker EEA1, and co-fractionates with a component of the AP5 complex. As the function of ZFYVE26 in neurons was largely unknown, we disrupted Zfyve26 in mice. Zfyve26 knockout mice do not show developmental defects but develop late-onset spastic paraplegia with cerebellar ataxia confirming that SPG15 is caused by ZFYVE26 deficiency. The morphological analysis reveals axon degeneration and progressive loss of both cortical motoneurons and Purkinje cells in the cerebellum. Importantly, neuron loss is preceded by accumulation of large intraneuronal deposits of membrane-surrounded material, which co-stains with the lysosomal marker Lamp1. A density gradient analysis of brain lysates shows an increase of Lamp1-positive membrane compartments with higher densities in Zfyve26 knockout mice. Increased levels of lysosomal enzymes in brains of aged knockout mice further support an alteration of the lysosomal compartment upon disruption of Zfyve26. We propose that SPG15 is caused by an endolysosomal membrane trafficking defect, which results in endolysosomal dysfunction. This appears to be particularly relevant in neurons with highly specialized neurites such as cortical motoneurons and Purkinje cells.     

Equivalence of regression curves sharing common parameters

In clinical trials, the comparison of two different populations is a common problem. Nonlinear (parametric) regression models are commonly used to describe the relationship between covariates, such as concentration or dose, and a response variable in the two groups. In some situations, it is reasonable to assume some model parameters to be the same, for instance, the placebo effect or the maximum treatment effect. In this paper, we develop a (parametric) bootstrap test to establish the similarity of two regression curves sharing some common parameters. We show by theoretical arguments and by means of a simulation study that the new test controls its significance level and achieves a reasonable power. Moreover, it is demonstrated that under the assumption of common parameters, a considerably more powerful test can be constructed compared with the test that does not use this assumption. Finally, we illustrate the potential applications of the new methodology by a clinical trial example.