Toll-like receptor 2-mediated innate immune response in human nonparenchymal liver cells toward adeno-associated viral vectors

Adeno-associated viral vectors (rAAV) are frequently used in gene therapy trials. Although rAAV vectors are of low immunogenicity, humoral as well as T cell responses may be induced. While the former limits vector reapplication, the expansion of cytotoxic T cells correlates with liver inflammation and loss of transduced hepatocytes. Because adaptive immune responses are a consequence of recognition by the innate immune system, we aimed to characterize cell autonomous immune responses elicited by rAAV in primary human hepatocytes and nonparenchymal liver cells. Surprisingly, Kupffer cells, but also liver sinusoidal endothelial cells, mounted responses to rAAV, whereas neither rAAV2 nor rAAV8 were recognized by hepatocytes. Viral capsids were sensed at the cell surface as pathogen-associated molecular patterns by Toll-like receptor 2. In contrast to the Toll-like receptor 9-mediated recognition observed in plasmacytoid dendritic cells, immune recognition of rAAV in primary human liver cells did not induce a type I interferon response, but up-regulated inflammatory cytokines through activation of nuclear factor κB. CONCLUSION: Using primary human liver cells, we identified a novel mechanism of rAAV recognition in the liver, demonstrating that alternative means of sensing rAAV particles have evolved. Minimizing this recognition will be key to improving rAAV-mediated gene transfer and reducing side effects in clinical trials due to immune responses against rAAV.     

Opposite effects of substance P and calcitonin gene-related peptide in oxazolone colitis

Background

Extrinsic sensory neurons play a crucial role in aberrant immune responses in colitis. The activation of peptidergic sensory nerve fibres is accompanied by a release of the neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP). SP levels increase whilst CGRP levels decrease in colon specimens from patients with inflammatory bowel disease; thus suggesting the pro- and anti-inflammatory roles, respectively, of these neuropeptides.

Methods

Oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one) colitis was induced in wild-type (WT), SP and CGRP knockout (^−/−) mice. CGRP^−/− mice were treated with the neurokinin 1-receptor antagonist CP-96345 (CP). The permeability of the mouse colon was evaluated by Evans Blue uptake. Cytokines produced by colonic lamina propria mononuclear cells were measured by ELISA.

Results

Colons of WT, CGRP^−/− and SP^−/− mice showed similar tissue architecture and permeability. SP^−/− mice were protected against oxazolone colitis, whereas CGRP^−/− showed increased susceptibility to colitis compared to WT mice. SP^−/− and CP-treated CGRP^−/− mice showed no significant body weight loss during the period of sickness in contrast to untreated CGRP^−/− and WT mice. Decreased production of IL-4, IL-5, and IL-13 by colonic lamina propria mononuclear cells of the protected SP^−/− mice confirms the crucial role of these cytokines in oxazolone colitis.

Conclusion

We demonstrate that the neuropeptides CGRP and SP exert opposing effects in oxazolone colitis and provide further evidence for a prominent neuroimmune association in the gut.     

Intestinal permeability in subjects from two different race groups with diverse stone-risk profiles

It is well established that calcium oxalate stones may be caused by colonic or ileum oxalate (Ox) hyperabsorption (secondary to intestinal dysfunction). Studies have reported that increased intestinal permeability (IP) can cause hyperabsorption of nutrients culminating in passive diffusion of Ox. In South Africa, renal stones occur in the white population (W) but are extremely rare in the black population (B). Previous studies have shown that despite B having a hyperoxalurogenic diet relative to W, urinary Ox in the former is not higher. It has been suggested that different Ox handling mechanisms in the groups are the cause of this disparity. The present study was undertaken to examine whether the IP index, a reliable and accurate measure of intestinal integrity, plays a role in this anomaly. Ten healthy males from each group ingested a dual-sugar isotonic solution containing 5 g lactulose (LA) and 2 g mannitol (MA). IP was assessed by comparing the LA:MA ratio in 5 h urine samples using high performance anion exchange chromatography coupled with pulse amperometric detection to measure the concentration of each sugar. 24 h dietary intake and urine composition were also determined. LA excretion was identical in both groups (0.03 %) while MA excretion was 8.3 % in B and 11.3 % in W. IP index was 0.004 for B and 0.003 for W. It is concluded that IP is not a contributory factor in the apparent different handling of dietary Ox in B and W South Africans. It is speculated that differences in renal transporters may play a role.     

Complication rates and outcomes stratified by treatment modalities in proximal humeral fractures: a systematic literature review from 1970–2009

Background

The optimal treatment of complex, displaced proximal humeral fractures is controversial. A systematic literature review of the time period from 1970 to 2009 was conducted. The purpose was to evaluate the clinical success and complications of the available treatment modalities to determine specific treatment recommendations for the different fracture patterns.

Methods

The databases (PubMed/EMBASE) were searched for the time period (01/1970–09/2009). Study quality, treatment modalities, classification, outcome scores and complications of 200 publications including 9377 patients were analyzed. Interventions were compared by analysis of variance with subsequent Tukey’s-test. Complication rates among methods were compared by using Pearson’s-chi-square-test and pairwise comparisons using Fisher’s-two-tailed-exact-test.

Results

Hemiarthroplasty, angle-stable plate and non-operative treatment were used for 63% of the follow-up-patients. For 3- and 4-part fractures, patients with hemiarthroplasty [3-Part: 56.4 (lower/upper 95% confidence interval (CI): 43.3-68.7); 4-Part: 49.4 (CI: 42.2-56.7)] received a lower score than different surgical head-preserving methods such as ORIF [3-Part: 82.4 (CI: 76.6-86.9); 4-Part: 83.0 (CI:78.7-86.6)], intramedullary nailing [3-Part: 79.1 (CI:74.0-83.4)] or angle-stable plates [4-Part: 66.4 (CI: 59.7-72.4)].The overall complication rate was 56%. The most common complications were fracture-displacement, malunion, humeral head necrosis and malreduction. The highest complication rates were documented for conventional plate and hemiarthroplasty and for AO-C, AO-A, for 3- and 4-part fractures. Only 25% of the data were reported with detailed classification results and the corresponding outcome scores.

Discussion

Despite the large amount of patients included, it is difficult to determine adequate recommendations for the treatment of proximal humeral fractures because a relevant lack of follow-up data impaired subsequent analysis. For displaced 3- and 4-part fractures head-preserving therapy received better outcome scores than hemiarthroplasty. However, a higher number of complications occurred in more complex fractures and when hemiarthroplasty or conventional plate osteosynthesis was performed. Thus, when informing the patient for consent, both the clinical results and the possibly expected complications with a chosen treatment modality should be addressed.

     

Maternal positive affect over the course of pregnancy is associated with the length of gestation and reduced risk of preterm delivery

Objective

The association between maternal psychological state during pregnancy and birth outcomes is well established. The focus of previous studies has been on the potentially detrimental consequences of maternal stress on pregnancy and birth outcomes, particularly shortened gestation and increased risk of preterm birth. Despite a growing literature linking positive affect with favorable health outcomes this construct has received little attention in the context of pregnancy. Therefore, in the current study, we tested the hypothesis that maternal positive affect during pregnancy is associated with beneficial consequences in terms of increased length of gestation and reduced risk of preterm birth above that of the absence of stress.

Methods

In 169 pregnant women maternal positive affect and perceived stress were serially assessed at 15.2±0.9 weeks (T1; mean ± SD), 19.7 ± 0.9 weeks (T2) and 30.7 ± 0.7 weeks (T3) gestation. Pregnancy and birth outcomes were abstracted from the medical record.

Results

Higher maternal positive affect and a steeper increase in maternal positive affect over pregnancy were positively associated with length of gestation (p < .05) and reduced risk of preterm delivery (p < .01), whereas maternal perceived stress was not significantly associated with shorter length of gestation (p > .10).

Conclusions

These findings suggest that maternal positive affect may be beneficial for outcomes related to the length gestation, and that this effect cannot be accounted for by the lower stress levels associated with higher positive affect. Interventions to increase maternal positive affect may be beneficial for fetal development.     

Validation of the German version of the extended ALS functional rating scale as a patient-reported outcome measure

The revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) is a well-established rating instrument to assess the functional status of ALS patients. A recent innovation was the addition of three further items designed to improve its sensitivity at lower levels of physical function (ALSFRS-Extension, ALSFRS-EX). Neither the ALSFRS-R nor the ALSFRS-EX has been validated in German yet. The aim of the present study was the validation of the German version of a self-administered form of the ALSFRS-EX. Seventy-six patients participated in the study. Psychometric analysis included reliability assessment and factorial analysis. To evaluate convergent validity, correlations between ALSFRS-EX items and the MRC score, spasticity, tongue movement, pulmonary function, ALSAQ-40 and Borg dyspnoea scales (upright and supine) were performed. Internal consistency as measured by Cronbach’s alpha (total scale 0.868, subscales 0.690–0.938) and corrected item to total correlations (all above 0.50) was high. Test–retest reliability assessed by Spearman’s rho (0.882–0.972) and Cohen’s Kappa (0.63–0.92) was also high. Principal component analysis with varimax rotation yielded a four-factor solution accounting for approximately 79 % of the variance. Clinical parameters were strongly correlated with respective items and subscores of the ALSFRS-EX (muscle strength 0.568–0.833 p < 0.01; spasticity ?0.236 to ?0.376 p < 0.05; tongue movement 0.437–0.818 p < 0.01; pulmonary function 0.485–0.577 p < 0.01). ALSAQ-40 and Borg score correlated highly with the corresponding ALSFRS-EX items. The German self-report version of the ALSFRS-EX possesses very good psychometric properties similar to the original scale including high internal consistency and test–retest reliability as well as excellent convergent validity.     

A role for vesicular glutamate transporter 1 in synaptic vesicle clustering and mobility

Synaptic vesicles (SVs) from excitatory synapses carry vesicular glutamate transporters (VGLUTs) that fill the vesicles with neurotransmitter. Although the essential function of VGLUTs as glutamate transporters has been well established, the evidence for additional cell‐biological functions is more controversial. Both VGLUT1 and VGLUT2 disruptions in mice result in a reduced number of SVs away from release sites, flattening of SVs, and the appearance of tubular structures. Therefore, we analysed the morphology, biochemical composition and trafficking of SVs at synapses of VGLUT1^?/? mice in order to test for a function of VGLUTs in the formation or clustering of SVs. Analyses with high‐pressure freezing immobilisation and electron tomography pointed to a role of VGLUT1 transport function in the tonicity of excitatory SVs, explaining the aldehyde‐induced flattening of SVs observed in VGLUT1^?/? synapses. We confirmed the steep reduction in the number of SVs previously observed in VGLUT1^?/? presynaptic terminals, but did not observe accumulation of endocytotic intermediates. Furthermore, SV proteins of adult VGLUT1^?/? mouse brain tissue were expressed at normal levels in all subcellular fractions, suggesting that they were not displaced to another organelle. We thus assessed the mobility of the recently documented superpool of SVs. Synaptobrevin2–enhanced green fluorescent protein time lapse experiments revealed an oversized superpool of SVs in VGLUT1^?/? neurons. Our results support the idea that, beyond glutamate loading, VGLUT1 enhances the tonicity of excitatory SVs and stabilises SVs at presynaptic terminals.